Phytases, nucleic acids encoding them and methods of making and using them

ABSTRACT

In one aspect, the invention provides a purified and modified phytase enzyme from  Escherichia coli  K12 appA phytase. The enzyme has phytase activity and improved thermal tolerance as compared with the wild-type enzyme. In addition, the enzyme has improved protease stability at low pH. Glycosylation of the modified phytase provided a further improved enzyme having improved thermal tolerance and protease stability. The enzyme can be produced from native or recombinant host cells and can be used to aid in the digestion of phytate where desired. In one aspect, the phytase of the present invention can be used in foodstuffs to improve the feeding value of phytate rich ingredients.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part (CIP) of U.S. patentapplication Ser. No. 09/866,379, filed May 24, 2001, which is acontinuation-in-part of U.S. patent application Ser. No. 09/580,515,filed May 25, 2000, which is a continuation-in-part of U.S. patentapplication Ser. No. 09/318,528, filed May 25, 1999, which is acontinuation-in-part of U.S. patent application Ser. No. 09/291,931,filed Apr. 13, 1999, which is a continuation of U.S. patent applicationSer. No. 09/259,214, filed Mar. 1, 1999, which is a divisional of U.S.patent application Ser. No. 08/910,798, now U.S. Pat. No. 5,876,997,filed Aug. 13, 1997, all of which are hereby incorporated by referencein their entirety for all purposes.

FIELD OF THE INVENTION

This invention relates to newly identified and newly engineeredpolynucleotides, polypeptides encoded by such polynucleotides, the useof such polynucleotides and polypeptides, as well as the production andisolation of such polynucleotides and polypeptides. More particularly,the polypeptides of the present invention have been identified asenzymes having phytase activity.

BACKGROUND OF THE INVENTION

Minerals are essential elements for the growth of all organisms. Dietaryminerals can be derived from many source materials, including plants.E.g., plant seeds are a rich source of minerals since they contain ionsthat are complexed with the phosphate groups of phytic acid molecules.These phytate-associated minerals satisfy the dietary needs of somespecies of farmed organisms, such as multi-stomached ruminants.Accordingly, ruminants do not require dietary supplementation withinorganic phosphate and minerals because microorganisms in the rumenproduce enzymes that catalyze conversion of phytate(myo-inositol-hexaphosphate) to inositol and inorganic phosphate. In theprocess, minerals that have been complexed with phytate are released.The majority of species of farmed organisms, however, are unable toefficiently utilize phytate-associated minerals. Thus, for example, inthe livestock production of monogastric animals (e.g., pigs, birds, andfish), feed is commonly supplemented with minerals &/or with antibioticsubstances that alter the digestive flora environment of the consumingorganism to enhance growth rates.

As such, there are many problematic burdens—related to nutrition, exvivo processing steps, health and medicine, environmental conservation,and resource management—that are associated with an insufficienthydrolysis of phytate in many applications. The following arenon-limiting examples of these problems:

-   -   1) The supplementation of diets with inorganic minerals is a        costly expense.    -   2) The presence of unhydrolyzed phytate is undesirable and        problematic in many ex vivo applications (e.g. by causing the        presence of unwanted sludge).    -   3) The supplementation of diets with antibiotics poses a medical        threat to humans and animals alike by increasing the abundance        of antibiotic-tolerant pathogens.    -   4) The discharge of unabsorbed fecal minerals into the        environment disrupts and damages the ecosystems of surrounding        soils, fish farm waters, and surface waters at large.    -   5) The valuable nutritional offerings of many potential        foodstuffs remain significantly untapped and squandered.

Many potentially nutritious plants, including particularly their seeds,contain appreciable amounts of nutrients, e.g. phosphate, that areassociated with phytate in a manner such that these nutrients are notfreely available upon consumption. The unavailability of these nutrientsis overcome by some organisms, including cows and other ruminants, thathave a sufficient digestive ability—largely derived from the presence ofsymbiotic life forms in their digestive tracts—to hydrolyze phytate andliberate the associated nutrients. However, the majority of species offarmed animals, including pigs, fish, chickens, turkeys, as well asother non-ruminant organisms including man, are unable to efficientlyliberate these nutrients after ingestion.

Consequently, phytate-containing foodstuffs require supplementation withexogenous nutrients and/or with a source of phytase activity in order toammend their deficient nutritional offerings upon consumption by a verylarge number of species of organisms.

In yet another aspect, the presence of unhydrolized phytate leads toproblematic consequences in ex vivo processes including—but not limitedto—the processing of foodstuffs. In but merely one exemplification, asdescribed in EP0321004-B1 (Vaara et al.), there is a step in theprocessing of corn and sorghum kernels whereby the hard kernels aresteeped in water to soften them. Water-soluble subtances that leach outduring this process become part of a corn steep liquor, which isconcentrated by evaporation. Unhydrolized phytic acid in the corn steepliquor, largely in the form of calcium and magnesium salts, isassociated with phosphorus and deposits an undesirable sludge withproteins and metal ions. This sludge is problematic in the evaporation,transportation and storage of the corn steep liquor. Accordingly, theinstantly disclosed phytase molecules—either alone or in combinationwith other reagents (including but not limited to enzymes, includingproteases)—are serviceable not only in this application (e.g., forprevention of the unwanted slugde) but also in other applications wherephytate hydrolysis is desirable.

The supplementation of diets with antibiotic substances has manybeneficial results in livestock production. For example, in addition toits role as a prophylactic means to ward off disease, the administrationof exogenous antibiotics has been shown to increase growth rates byupwards of 3-5%. The mechanism of this action may also involve—inpart—an alteration in the digestive flora environment of farmed animals,resulting in a microfloral balance that is more optimal for nutrientabsorption.

However, a significant negative effect associated with the overuse ofantibiotics is the danger of creating a repository of pathogenicantibiotic-resistant microbial strains. This danger is imminent, and therise of drug-resistant pathogens in humans has already been linked tothe use of antibiotics in livestock. For example, Avoparcin, theantibiotic used in animal feeds, was banned in many places in 1997, andanimals are now being given another antibiotic, virginiamycin, which isvery similar to the new drug, Synercid®, used to replace vancomycin inhuman beings. However, studies have already shown that some enterococciin farm animals are resistant to Synercid®. Consequently, undesiredtolerance consequences, such as those already seen with Avoparcin andvancomycin, are likely to reoccur no matter what new antibiotics areused as blanket prophylactics for farmed animals. Accordingly,researchers are calling for tighter controls on drug use in theindustry.

The increases in growth rates achieved in animals raised on foodstuffssupplemented with the instantly disclosed phytase molecules matches—ifnot exceeds—those achieved using antibiotics such as, for example,Avoparcin. Accordingly, the instantly disclosed phytase molecules—eitheralone or in combination with other reagents (including but not limitedto enzymes, including proteases)—are serviceable not only in thisapplication (e.g., for increasing the growth rate of farmed animals) butalso in other applications where phytate hydrolysis is desirable.

An environmental consequence is that the consumption ofphytate-containing foodstuffs by any organism species that isphytase-deficient—regardless of whether the foodstuffs are supplementedwith minerals—leads to fecal pollution resulting from the excretion ofunabsorbed minerals. This pollution has a negative impact not only onthe immediate habitat but consequently also on the surrounding waters.The environmental alterations occur primarily at the bottom of the foodchain, and therefore have the potential to permeate upwards andthroughout an ecosystem to effect permanent and catastrophicdamage—particularly after years of continual pollution. This problem hasthe potential to manifest itself in any area where concentrated phytateprocessing occurs—including in vivo (e.g. by animals in areas oflivestock production, zoological grounds, wildlife refuges, etc.) and invitro (e.g. in commercial corn wet milling, ceral steeping processes,etc.) processing steps.

The decision to use exogenously added phytase molecules—whether to fullyreplace or to augment the use of exogenously administered minerals &/orantibiotics—ultimately needs to pass a test of financial feasibility &cost effectiveness by the user whose livelihood depends on the relevantapplication, such as livestock production.

Consequently, there is a need for means to achieve efficient and costeffective hydrolysis of phytate in various applications. Particularly,there is a need for means to optimize the hyrolysis of phytate incommercial applications. In a particular aspect, there is a need tooptimize commercial treatment methods that improve the nutritionalofferings of phytate-containing foodstuffs for consumption by humans andfarmed animals.

Previous reports of recombinant phytases are available, but theirinferior activities are eclipsed by the newly discovered phytasemolecules of instant invention. Accordingly, the instantly disclosedphytase molecules are counted upon to provide substantially superiorcommercial performance than previously identified phytase molecules,e.g. phytase molecules of fungal origin.

Phytate occurs as a source of stored phosphorous in virtually all plantfeeds (Graf (Ed.), 1986). Phytic acid forms a normal part of the seed incereals and legumes. It functions to bind dietary minerals that areessential to the new plant as it emerges from the seed. When thephosphate groups of phytic acid are removed by the seed enzyme phytase,the ability to bind metal ions is lost and the minerals become availableto the plant. In livestock feed grains, the trace minerals bound byphytic acid are largely unavailable for absorption by monogastricanimals, which lack phytase activity.

Although some hydrolysis of phytate occurs in the colon, most phytatepasses through the gastrointestinal tract of monogastric animals and isexcreted in the manure contributing to fecal phosphate pollutionproblems in areas of intense livestock production. Inorganic phosphorousreleased in the colon has an appreciably diminished nutritional value tolivestock because inorganic phosphorous is absorbed mostly—if notvirtually exclusively—in the small intestine. Thus, an appreciableamount of the nutritionally important dietary minerals in phytate isunavailable to monogastric animals.

In sum, phytate-associated nutrients are comprised of not only phosphatethat is covalently linked to phytate, but also other minerals that arechelated by phytate as well. Moreover, upon injestion, unhydrolyzedphytate may further encounter and become associated with additionalminerals. The chelation of minerals may inhibit the activity of enzymesfor which these minerals serve as co-factors.

Conversion of phytate to inositol and inorganic phosphorous can becatalyzed by microbial enzymes referred to broadly as phytases. Phytasessuch as phytase #EC 3.1.3.8 are capable of catalyzing the hydrolysis ofmyo-inositol hexaphosphate to D-myo-inositol 1,2,4,5,6-pentaphosphateand orthophosphate. Certain fungal phytases reportedly hydrolyzeinositol pentaphosphate to tetra-, tri-, and lower phosphates. E.g., A.ficuum phytases reportedly produce mixtures of myoinositol di- andmono-phosphates (Ullah, 1988). Phytase-producing microorganisms arecomprised of bacteria such as Bacillus subtilis (Powar and Jagannathan,1982) and Pseudomonas (Cosgrove, 1970); yeasts such as Sacchoromycescerevisiae (Nayini and Markakis, 1984); and fungi such as Aspergillusterreus (Yamada et al., 1968).

Acid phosphatases are enzymes that catalytically hydrolyze a widevariety of phosphate esters and usually exhibit pH optima below 6.0(Igarashi & Hollander, 1968). E.g., #EC 3.1.3.2 enzymes catalyze thehydrolysis of orthophosphoric monoesters to orthophosphate products. Anacid phosphatase has reportedly been purified from A. ficuum. Thedeglycosylated form of the acid phosphatase has an apparent molecularweight of 32.6 kDa (Ullah et al., 1987).

Phytase and less specific acid phosphatases are produced by the fungusAspergillus ficuum as extracellular enzymes (Shieh et al., 1969). Ullahreportedly purified a phytase from wild-type A. ficuum that had anapparent molecular weight of 61.7 kDA (on SDS-PAGE; as corrected forglycosylation); pH optima at pH 2.5 and pH 5.5; a Km of about 401 μm;and, a specific activity of about 50 U/mg (Ullah, 1988). PCT patentapplication WO 91/05053 also reportedly discloses isolation andmolecular cloning of a phytase from Aspergillus ficuum with pH optima atpH 2.5 and pH 5.5, a Km of about 250 μm, and specific activity of about100 U/mg protein.

Summarily, the specific activity cited for these previously reportedmicrobial enzymes has been approximately in the range of 50-100 U/mgprotein. In contrast, the phytase activity disclosed in the instantinvention has been measured to be approximately 4400 U/mg. Thiscorresponds to about a 40-fold or better improvement in activity.

The possibility of using microbes capable of producing phytase as a feedadditive for monogastric animals has been reported previously (U.S. Pat.No. 3,297,548 Shieh and Ware; Nelson et al., 1971). Thecost-effectiveness of this approach has been a major limitation for thisand other commercial applications. Therefore improved phytase moleculesare highly desirable.

Microbial phytases may also reportedly be useful for producing animalfeed from certain industrial processes, e.g., wheat and corn wasteproducts. In one aspect, the wet milling process of corn producesglutens sold as animal feeds. The addition of phytase may reportedlyimprove the nutritional value of the feed product. For example, the useof fungal phytase enzymes and process conditions (t˜50° C. and pH 5.5)have been reported previously in (e.g. EP 0 321 004). Briefly, inprocessing soybean meal using traditional steeping methods, i.e.,methods without the addition of exogenous phytase enzyme, the presenceof unhydrolyzed phytate reportedly renders the meal and wastesunsuitable for feeds used in rearing fish, poultry and othernon-ruminants as well as calves fed on milk. Phytase is reportedlyuseful for improving the nutrient and commercial value of this highprotein soy material (see Finase Enzymes by Alko, Rajamäki, Finland). Acombination of fungal phytase and a pH 2.5 optimum acid phosphatase formA. niger has been used by Alko, Ltd as an animal feed supplement intheir phytic acid degradative product Finas F and Finase S. However, thecost-effectiveness of this approach has remained a major limitation tomore widespread use. Thus a cost-effective source of phytase wouldgreatly enhance the value of soybean meals as an animal feed (Shieh etal., 1969).

To solve the problems disclosed, the treatment of foodstuffs withexogenous phytase enzymes has been proposed, but this approach was notbeen fully optimized, particularly with respect to feasibility and costefficiency. This optimization requires the consideration that a widerange of applications exists, particularly for large scale production.For example, there is a wide range of foodstuffs, preparation methodsthereof, and species of recipient organisms.

In a particular exemplification, it is appreciated that the manufactureof fish feed pellets requires exposure of ingedients to hightemperatures &/or pressure in order to produce pellets that do notdissolve &/or degrade prematurely (e.g. e.g. prior to consumption) uponsubjection to water. It would thus be desirable for this manufacturingprocess to obtain additive enzymes that are stable under hightemperature and/or pressure conditions. Accordingly it is appreciatedthat distinct phytases may be differentially preferable or optimal fordistinct applications.

In sum, there is a need for novel, highly active, physiologicallyeffective, and economical sources of phytase activity. Specifically,there is a need to identify novel phytases that: a) have superioractivities under one or more specific applications, and are thusserviceable for optimizing these specific applications; b) areserviceable as templates for directed evolution to achieve even furtherimproved novel molecules; and c) are serviceable as tools for theidentification of additional related molecules by means such ashybridization-based approaches.

SUMMARY OF THE INVENTION

The invention provides a formulation or a pharmaceutical compositioncomprising at least one polypeptide having phytase activity, wherein thepolypeptide comprises: (a) a polypeptide encoded by a nucleic acidcomprising a nucleotide sequence as set forth in SEQ ID NO:7 and whereinnucleotide 389 is G; 390 is A; 437 is T; 438 is G; 439 is G; 470 is C;472 is T; 476 is T; 477 is G; 478 is T; 689 is G; 690 is A; 691 is G;728 is T; 729 is A; 730 is T; 863 is T; 864 is G; or, 1016 is G; or, anycombination thereof, wherein the polynucleotide encodes a phytase; (b) apolypeptide encoded by a nucleic acid comprising a nucleotide sequenceas set forth in SEQ ID NO:7 and wherein nucleotide 389 is G; 390 is A;437 is T; 438 is G; 439 is G; 470 is C; 472 is T; 476 is T; 477 is G;478 is T; 689 is G; 690 is A; 691 is G; 728 is T; 729 is A; 730 is T;863 is T; 864 is G; and 1016 is G; (c) a polypeptide having an aminoacid sequence as set forth in SEQ ID NO:8 and having one or more aminoacid modifications selected from W68E, Q84W, A95P, K97C, S168E, R181Y,N226C, Y277D or any combination thereof, wherein the polypeptide hasphytase activity; (d) a polypeptide having an amino acid sequence as setforth in SEQ ID NO:8 and having the amino acid modifications W68E, Q84W,A95P, K97C, S1168E, R181 Y, N226C, Y277D, wherein the polypeptide hasphytase activity; (e) a polypeptide encoded by a nucleic acid comprisinga nucleotide sequence as set forth in SEQ ID NO:1; (f) a polypeptidehaving an amino acid sequence as set forth in SEQ ID NO:2; or, (g) acombination of (a), (b), (c), (d), (e) or (f).

In one aspect, the formulation is a dietary supplement, or, apharmaceutical composition. In one aspect, the formulation orpharmaceutical composition further comprises at least one vitamin, atleast one additional enzyme, at least one mineral or metal, or at leastone herb or plant extract, at least one amino acid or amino acidderivative, or any combination thereof. The mineral or metal can bealuminum, antimony, barium, beryllium, bismuth, boron, bromide, bromine,cadmium, calcium, cerium, cesium, chloride, chromium, cobalt, copper,dysprosium, erbium, europium, fluoride, fluorine, gadolinium, gallium,germanium, gold, hafnium, holmium, indium, iodine, iridium, iron,lanthanum, lithium, lutetium, magnesium, manganese, molybdenum,neodymium, nickel, niobium, osmium, palladium, phosphorous, platinum,potassium, praseodymium, promethium, rhenium, rhodium, rubidium,ruthenium, samarium, scandium, selenium, silicon, silver, sodium,strontium, sulfur, tantalum, tellurium, terbium, thorium, thulium, tin,titanium, tungsten, vanadium, xinconium, ytterbium, yttrium, zinc,zirconium or any combination thereof. In one aspect, the formulation orpharmaceutical composition further comprises a composition comprising adiatomaceous earth, charcoal, choline, inositol, biotin, PABA,Alpha-Lipoic Acid, a carotenoid, beta carotene, coenzyme Q10,chondroitin, melatonin, lecithin, brewer's yeast or a combinationthereof. The herb or plant extract can comprise an alfalfa, a ginseng,American ginseng, Asian red ginseng, Asian white ginseng, Siberianginseng, Brazilian ginseng, astragalus, bilberry, black cohosh, cascarasagrada, cat's claw, cayenne, dong quai, echinacea, eucalyptus,feverfew, garlic, ginkgo biloba, goldenseal, gotu kola, horsetail, maca,a mushroom, Maitake mushroom, Reishi mushroom, Shuitake mushroom,leuzea, rhodiola, milk thistle, noni, pau d'arco, papaya, pygeum, sawpalmetto, schizandra, senna, suma, wild yam, willow, yucca, wheat grass,barley grass, parsley, broccoli, acerola cherries, aloe vera, quercitin,pine bark, grape seed, green tea, red wine, grapefruit extract, ginger,oat straw, sarsaparilla, an oil, walnut oil, safflower oil, soybean oil,peanut oil, a fish oil, salmon oil, evening primrose oil, borage oil,bee pollen, bee propolis, royal jelly, a bran, oat bran, wheat bran, afiber, soy, psyllium, apple pectin, a protein, egg protein, milkprotein, soy protein, rice protein, whey, algae, spirulina, Chlorella,dulse, kelp, D. salina or a combination thereof. The probiotic cancomprise a Lactobacillus species, L. acidophilus,. L. bifidus, L.sporogenes, L. casei, L. rhamnosus, L. plantarum, S. thermophilus, aBifidobacterium species, an Escherichia, an Enterococcus, a Bacillus ora Saccharomyces species. The additional enzyme can be a phytase, anamylase, a bromelain, a cellulase, a chymopapain, a diastase, aglucoamylase, a hemicellulase, a hyaluronidase, an invertase, a lactase,a lipase, a maltase, a pancreatin, a papain, a pectinase, a pepsin, aplasmin, a protease, a rennin or any combination thereof. The vitamincan comprise vitamin B, Thiamine (Vitamin B1), Riboflavin (Vitamin B2),Nicotinic acid (Niacin, Vitamin B3), Pantothenic acid (Vitamin B5),Pyridoxine (Vitamin B6), B7, Folic acid (Vitamin B9), Cyanocobalamin(Vitamin B12), vitamin C, a vitamin D, vitamin D1, vitamin D2, vitaminD3, vitamin E, a vitamin K, vitamin K1, vitamin K2, vitamin G, vitaminH, vitamin P or any combination thereof. The amino acid or amino acidderivative can comprise Isoleucine, Leucine, Lysine, Phenylalanine,Threonine, Tryptophan, Valine, Methionine, Cysteine, Alanine, Arginine,Aspartic Acid, Glutamic Acid, Glycine, Histidine, Proline, Serine,Asparagine, Glutamine, Tyrosine, taurine, glucosamine or any combinationthereof. In one aspect, the formulation or pharmaceutical compositionfurther comprises vitamin D3 or calcium or both. In one aspect, theformulation or pharmaceutical composition further comprises potassium,glucose, CaCl₂ or a combination thereof. In one aspect, the formulationor pharmaceutical composition further comprises at least one enzymeselected from the group consisting of α-galactosidases,β-galactosidases, lactases, phytases, β-glucanases,endo-β-1,4-glucanases and endo-β-1,3(4)-glucanases, cellulases,xylosidases, galactanases, arabinogalactan endo-1,4-β-galactosidases andarabinogalactan endo-1,3-β-galactosidases, endoglucanases,endo-1,2-β-glucanase, endo-1,3-α-glucanase, endo-1,3-β-glucanase, pectindegrading enzymes, pectinases, pectinesterases, pectin lyases,polygalacturonases, arabinanases, rhamnogalacturonases,rhamnogalacturonan acetyl esterases, rhamnogalacturonan-α-rhamnosidase,pectate lyases, α-galacturonisidases, mannanases, β-mannosidases, mannanacetyl esterases, xylan acetyl esterases, proteases, xylanases,arabinoxylanases, lipases, phospholipases or a cutinase. In one aspect,the formulation or pharmaceutical composition comprises a powder, atablet, a concentrate, a geltab, a capsule, a spray, an aerosol, alotion, an adhesive patch or a drink.

The invention provides pharmaceutical composition comprising at leastone polypeptide having phytase activity and a pharmaceuticallyacceptable excipient, wherein the polypeptide comprises: (a) apolypeptide encoded by a nucleic acid comprising a nucleotide sequenceas set forth in SEQ ID NO:7 and wherein nucleotide 389 is G; 390 is A;437 is T; 438 is G; 439 is G; 470 is C; 472 is T; 476 is T; 477 is G;478 is T; 689 is G; 690 is A; 691 is G; 728 is T; 729 is A; 730 is T;863 is T; 864 is G; or, 1016 is G; or, any combination thereof, whereinthe polynucleotide encodes a phytase; (b) a polypeptide encoded by anucleic acid comprising a nucleotide sequence as set forth in SEQ IDNO:7 and wherein nucleotide 389 is G; 390 is A; 437 is T; 438 is G; 439is G; 470 is C; 472 is T; 476 is T; 477 is G; 478 is T; 689 is G; 690 isA; 691 is G; 728 is T; 729 is A; 730 is T; 863 is T; 864 is G; and 1016is G; (c) a polypeptide having an amino acid sequence as set forth inSEQ ID NO:8 and having one or more amino acid modifications selectedfrom W68E, Q84W, A95P, K97C, S168E, R181 Y, N226C, Y277D or anycombination thereof, wherein the polypeptide has phytase activity; (d) apolypeptide having an amino acid sequence as set forth in SEQ ID NO:8and having the amino acid modifications W68E, Q84W, A95P, K97C, S168E,R181Y, N226C, Y277D, wherein the polypeptide has phytase activity; (e) apolypeptide encoded by a nucleic acid comprising a nucleotide sequenceas set forth in SEQ ID NO:1; (f) a polypeptide having an amino acidsequence as set forth in SEQ ID NO:2; or, (g) a combination of (a), (b),(c), (d), (e) or (f). In one aspect, the formulation or pharmaceuticalcomposition is formulated for oral delivery. In one aspect, theformulation or pharmaceutical composition is formulated as a pill, atablet, a capsule, a spray, an aerosol or a powder.

The invention provides a kit comprising a formulation of the invention,or a pharmaceutical composition of the invention, and instructions onusing the formulation or the pharmaceutical composition.

The invention provides immobilized phytases comprising: (a) apolypeptide encoded by a nucleic acid comprising a nucleotide sequenceas set forth in SEQ ID NO:7 and wherein nucleotide 389 is G; 390 is A;437 is T; 438 is G; 439 is G; 470 is C; 472 is T; 476 is T; 477 is G;478 is T; 689 is G; 690 is A; 691 is G; 728 is T; 729 is A; 730 is T;863 is T; 864 is G; or, 1016 is G; or, any combination thereof, whereinthe polynucleotide encodes a phytase; (b) a polypeptide encoded by anucleic acid comprising a nucleotide sequence as set forth in SEQ IDNO:7 and wherein nucleotide 389 is G; 390 is A; 437 is T; 438 is G; 439is G; 470 is C; 472 is T; 476 is T; 477 is G; 478 is T; 689 is G; 690 isA; 691 is G; 728 is T; 729 is A; 730 is T; 863 is T; 864 is G; and 1016is G; (c) a polypeptide having an amino acid sequence as set forth inSEQ ID NO:8 and having one or more amino acid modifications selectedfrom W68E, Q84W, A95P, K97C, S168E, R181Y, N226C, Y277D or anycombination thereof, wherein the polypeptide has phytase activity; (d) apolypeptide having an amino acid sequence as set forth in SEQ ID NO:8and having the amino acid modifications W68E, Q84W, A95P, K97C, S168E,R181Y, N226C, Y277D, wherein the polypeptide has phytase activity; (e) apolypeptide encoded by a nucleic acid comprising a nucleotide sequenceas set forth in SEQ ID NO:1; (f) a polypeptide having an amino acidsequence as set forth in SEQ ID NO:2; or, (g) a combination of (a), (b),(c), (d), (e) or (f). In one aspect, the polypeptide is immobilized to abead, for example, a polysorb bead or a polystyrene bead, or equivalentbead.

The invention provides a dietary supplement comprising the immobilizedphytase of the invention. In one aspect, the invention provides apharmaceutical composition comprising the immobilized phytase of theinvention.

The invention provides fertilizer or soil additives comprising at leastone immobilized phytase of the invention. The invention providesfertilizer or soil additives comprising at least one polypeptide of theinvention having phytase activity. The invention provides liquidsupplements for preventing muscle cramps comprising a formulation of theinvention or a pharmaceutical composition of the invention. In oneaspect, the formulation or pharmaceutical composition can furthercomprise glucose, potassium, sodium or calcium.

The invention provides hydrating agents comprising a formulation orpharmaceutical composition of the invention. The hydrating agents canfurther comprise glucose, potassium, sodium or calcium.

The invention provides tissue culture or cell culture media or cellculture media additive comprising at least one polypeptide of theinvention having phytase activity.

The invention provides plant food additives comprising at least onepolypeptide of the invention having phytase activity.

The invention provides methods for treating or preventing osteoporosisin an individual comprising administering to an individual an effectiveamount of a formulation or pharmaceutical composition of the invention.

The invention provides method for treating or preventing bone loss in anindividual comprising administering to an individual an effective amountof a formulation or pharmaceutical composition of the invention.

The invention provides methods for reversing bone loss or osteoporosisin an individual comprising administering to an individual an effectiveamount of a formulation or pharmaceutical composition of the invention.

The invention provides methods for preventing muscle cramps comprisingadministering to an individual an effective amount of a formulation orpharmaceutical composition of the invention.

The invention provides methods for reducing pollution and increasingnutrient availability in an environment or environmental sample bydegrading environmental phytic acid comprising applying to theenvironmental or environmental sample an effective amount of acomposition comprising at least one polypeptide of the invention havingphytase activity. In one aspect of the methods, the environment orenvironmental sample comprises a soil or a body of water, for example, awell, a pond, a lake, a river, an aquifer or a reservoir. In one aspectof the methods, the environment or environmental sample comprises asewage, a sewage effluent, a landfill or a manure pond.

The present invention provides a polynucleotide and a polypeptideencoded thereby which has been identified as a phytase enzyme havingphytase activity. In accordance with one aspect of the presentinvention, there is provided a novel recombinant enzyme, as well asactive fragments, analogs and derivatives thereof.

A deposit of the gene designated 819PH59 in E. coli XL1-Blue has beenmade with and accepted by the ATCC located at 10801 University Blvd.,Manassas, Va. 20110-2209, on Nov. 26, 2002. The Patent DepositDesignation is PTA-4822.

In one aspect, this invention relates to the use of recombinant phytasemolecules of bacterial origin that are serviceable for improving thenutritional value of phytate-containing foodstuffs. Previouspublications have disclosed the use of fungal phytases, but the use ofbacterial phyatases for this purpose is novel.

In one aspect, this invention relates to the use of newly identifiedrecombinant phytase molecules of E. coli origin that are serviceable forimproving the nutritional value of phytate-containing foodstuffs.

This use is comprised of employing the newly identified molecules tohydrolyze phytate in foodstuffs. Hydrolysis may occur before injestionor after injestion or both before and after injestion of the phytate.This application is particularly relevant, but not limited, tonon-ruminant organisms and includes the expression of the disclosednovel phytase molecules in transformed hosts, the contacting of thedisclosed novel phytase molecules with phytate in foodstuffs and othermaterials, and the treatment of animal digestive systems with thedisclosed novel phytase molecules.

Additionally, hydrolysis may occur independently of consumption, e.g. inan in vitro application, such as in a reaction vessel. Thus, thetreatment of phytate-containing materials includes the treatment of awide range of materials, including ones that are not intended to befoodstuffs, e.g. the treatment of excrementary (or fecal) material.

In one aspect, molecules of the present invention include a recombinantphytase isolated from Escherichia coli B that improves the efficiency ofrelease of phosphorous from phytate and the salts of phytic acid whencompared to previosuly identified fungal phytases.

In one aspect, there is provided a phytase enzyme that is serviceablefor incorportion into foodstuffs. In one aspect, there is provided aphytase enzyme that is serviceable for improving the nutritional valueof phytate-containing foodstuffs. More specifically still, there isprovided a phytase enzyme that, when applied to phytate-containingfoodstuffs, measurably improves the growth performance of an organismthat consumes it. It is theorized that the beneficial mechanism ofaction of the phytase activity is comprised appreciably if notsubstantially of the hydrolysis of phytate. It is provided that thebeneficial action may occur before injestion or alternatively afterinjestion or alternatively both before and after injestion of thephytate-containing foodstuff. In the case where the beneficial actionoccurs after injestion, it is an object of the present invention toprovide a phytase enzyme that has activity that is retained uponconsumption by non-ruminant organisms.

In one aspect, there are provided isolated nucleic acid moleculesencoding the enzyme of the present invention—including mRNA, DNA, cDNA,genomic DNA—as well as active derivatives, analogs and fragments of suchenzyme.

In one aspect, there is provided a process for producing suchpolypeptides by recombinant techniques comprising culturing recombinantprokaryotic and/or eukaryotic host cells, containing a nucleic acidsequence encoding an enzyme of the present invention, under conditionspromoting expression of the enzyme and subsequent recovery of theenzyme.

In one aspect, there is provided a process for expressing such enzymes,or polynucleotides encoding such enzymes in transgenic plants or plantorgans and methods for the production of such plants. This is achievableby introducing into a plant an expression construct comprised of anucleic acid sequence encoding such phytase enzymes.

In one aspect, there is provided a process for utilizing such enzymes,or polynucleotides encoding such enzymes for use in commercialprocesses, such as, for example, processes that liberate minerals fromphytates in plant materials either in vitro, i.e., in feed treatmentprocesses, or in vivo, i.e., by administering the enzymes to animals.

In one aspect, there are provided foodstuffs made by the disclosed feedtreatment processes.

In accordance with yet a further aspect of the present invention, thereare provided a processes for utilizing such enzymes, or polynucleotidesencoding such enzymes, for in vitro purposes related to research,discovery, and development. In a non-limiting exemplification, suchprocesses comprise the generation of probes for identifying andisolating similar sequences which might encode similar enzymes fromother organisms.

In a particular non-limiting exemplification, there are also providedprocesses for generating nucleic acid probes comprising nucleic acidmolecules of sufficient length to specifically hybridize to a nucleicacid sequence of the present invention. By way of preferredexemplification, hybridization-based uses of these probes include, butare by no means limited to, PCR, Northern and Southern types ofhybridizations, RNA protection assays, and in situ types ofhybridizations. The uses of the instantly disclosed molecules furtherinclude, in a non-limiting manner, diagnostic applications.

In accordance with a non-limiting exemplification, these processescomprise the generation of antibodies to the disclosed molecules, anduses of such antibodies, including, for example, for the identificationand isolation of similar sequences in enzymes from other organisms. Inanother non-limiting examplification, these processes include the use ofthe present enzymes as templates for directed evolution, comprising thegeneration of novel molecules by followed by screening-based approachesfor discoverying of progeny molecules with improved properties.

Also provided is a transgenic non-human organism whose genome comprisesa heterologous nucleic acid sequence encoding a polypeptide havingphytase activity, wherein the transgene results in expression of aphytase polypeptide.

The invention also provides phytase encoding polynucleotides having anucleotide sequence substantially identical to SEQ ID NO:7, and having amodified nucleotide sequence selected from nucleotide 389 is G; 390 isA; nucleotide 437 is T; 438 is G; 439 is G; 470 is C; 472 is T; 476 isT; 477 is G; 478 is T; 689 is G; 690 is A; 691 is G; 728 is T; 729 is A;730 is T; 863 is T; 864 is G; 1016 is G, or any combination thereof.Further, the invention provides a polynucleotide having a nucleotidesequence substantially identical to SEQ ID NO:7, and having a modifiednucleotide sequence selected from nucleotide 389 is G and 390 is A;nucleotide 437 is T, 438 is G and 439 is G; 470 is C and 472 is T; 476is T, 477 is G, and 478 is T; 689 is G, 690 is A and 691 is G; 728 is T,729 is A, and 730 is T; 863 is T and 864 is G; 1016 is G, or anycombination thereof. The later sequence is exemplified in SEQ ID NO:9and the corresponding amino acid sequence is SEQ ID NO:10.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIGS. 1 a and 1 b show the nucleotide and deduced amino acid sequencesan exemplary enzyme of the present invention. Sequencing was performedusing a 378 automated DNA sequencer (Applied Biosystems, Inc.).

FIGS. 2A and 2B show the pH and temperature profile and stability datafor the phytase enzyme of the present invention, as described in detailin Example 6, below.

FIG. 3 shows a graph with the results of a thermal tolerance assaybetween SEQ ID NO:8 (E. coli appA wild type) and an exemplary phytase ofthe invention having a sequence as set forth in SEQ ID NO:10 (modifiedphytase).

FIG. 4 shows a graph with the stability of phytase enzymes undersimulated digestibility conditions.

FIG. 5 shows a graph with expression of wild-type and modified phytase(SEQ ID NO:10) in various host cells.

FIG. 6 shows a graph of residual phytase activity in SGF with pepsin.

FIGS. 7A and 7B show the nucleotide sequence of E. coli appA phytase(SEQ ID NO:7, encoding the E. coli appA wild type phytase SEQ ID NO:8).

FIG. 8 shows the amino acid sequence of E. coli appA phytase (SEQ IDNO:8) and an exemplary phytase of the invention having a sequence as setforth in SEQ ID NO:10 (a modified phytase).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a thermally stable phytase having apolynucleotide and polypeptide sequence modified in such a way as toprovide a phytase havng increased thermal stability as compared withother phytase enzymes. Expression of this new phytase in S. pombe or P.pastoris, for example, resulted in the production of glycosylatedvariants that exhibited additional thermal tolerance.

The present invention provides purified a recombinant phytase enzyme,shown in FIGS. 1 a and 1 b. Additionally, the present invention providesisolated nucleic acid molecules (polynucleotides) which encode for themature enzyme having the deduced amino acid sequence of FIGS. 1 a and 1b. The invention also provides an exemplary phytase of the inventionhaving a sequence as set forth in SEQ ID NO:10, and encoded by, e.g.,SEQ ID NO:9 (a modified phytase sequence) as shown in FIG. 8 and SEQ IDNO:9 and 10.

The phytase molecules of the instant invention are novel with respect totheir structures. Additionally, the instant phytase molecules arepatentably novel with respect to activity. For example, using an assay(as described in Food Chemicals Codex, 4^(th) Ed.) the activity of theinstant phytase enzyme was demonstrated to be far superior in comparisonto a fungal (Aspergillus) phytase control. Specifically, a plurality ofexperiments showed the E. coli phytase to have an activity of about 4400units/mg and the Aspergillus phytase to have an activity of about 105units/mg. This corresponds to more than a 40-fold difference inactivity. In order to facilitate understanding of the examples providedherein, certain frequently occurring methods and/or terms will bedescribed.

The term “antibody,” as used herein, refers to intact immunoglobulinmolecules, as well as fragments of immunoglobulin molecules, such asFab, Fab′, (Fab′)₂, Fv, and SCA fragments, that are capable of bindingto an epitope of a phytase polypeptide. These antibody fragments, whichretain some ability to selectively bind to the antigen (e.g., an phytaseantigen) of the antibody from which they are derived, can be made usingwell known methods in the art (see, e.g., Harlow and Lane, supra), andare described further, as follows.

-   -   (1) An Fab fragment consists of a monovalent antigen-binding        fragment of an antibody molecule, and can be produced by        digestion of a whole antibody molecule with the enzyme papain,        to yield a fragment consisting of an intact light chain and a        portion of a heavy chain.    -   (2) An Fab′ fragment of an antibody molecule can be obtained by        treating a whole antibody molecule with pepsin, followed by        reduction, to yield a molecule consisting of an intact light        chain and a portion of a heavy chain. Two Fab′ fragments are        obtained per antibody molecule treated in this manner.    -   (3) An (Fab′)₂ fragment of an antibody can be obtained by        treating a whole antibody molecule with the enzyme pepsin,        without subsequent reduction. A (Fab′)₂ fragment is a dimer of        two Fab′ fragments, held together by two disulfide bonds.    -   (4) An Fv fragment is defined as a genetically engineered        fragment containing the variable region of a light chain and the        variable region of a heavy chain expressed as two chains.    -   (5) An single chain antibody (“SCA”) is a genetically engineered        single chain molecule containing the variable region of a light        chain and the variable region of a heavy chain, linked by a        suitable, flexible polypeptide linker.

The term “degrading effective” amount refers to the amount of enzymewhich is required to degrade at least 50% of the phytate, as compared tophytate not contacted with the enzyme. In one aspect, at least 80% ofthe phytate is degraded.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a gel to isolate the desired fragment.

As used in this invention, the term “epitope” refers to an antigenicdeterminant on an antigen, such as a phytase polypeptide, to which theparatope of an antibody, such as an phytase-specific antibody, binds.Antigenic determinants usually consist of chemically active surfacegroupings of molecules, such as amino acids or sugar side chains, andcan have specific three-dimensional structural characteristics, as wellas specific charge characteristics.

The terms “fragment”, “derivative” and “analog” when referring to theenzyme of FIGS. 1 a and 1 b comprise a enzyme which retains at least onebiological function or activity that is at least essentially same asthat of the reference enzyme. Furthermore, the terms “fragment”,“derivative” or “analog” are exemplified by a “pro-form” molecule, suchas a low activity proprotein that can be modified by cleavage to producea mature enzyme with significantly higher activity.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide or enzymepresent in a living animal is not isolated, but the same polynucleotideor enzyme, separated from some or all of the coexisting materials in thenatural system, is isolated. Such polynucleotides could be part of avector and/or such polynucleotides or enzymes could be part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment.

By “isolated nucleic acid” is meant a nucleic acid, e.g., a DNA or RNAmolecule, that is not immediately contiguous with the 5′ and 3′ flankingsequences with which it normally is immediately contiguous when presentin the naturally occurring genome of the organism from which it isderived. The term thus describes, for example, a nucleic acid that isincorporated into a vector, such as a plasmid or viral vector; a nucleicacid that is incorporated into the genome of a heterologous cell (or thegenome of a homologous cell, but at a site different from that at whichit naturally occurs); and a nucleic acid that exists as a separatemolecule, e.g., a DNA fragment produced by PCR amplification orrestriction enzyme digestion, or an RNA molecule produced by in vitrotranscription. The term also describes a recombinant nucleic acid thatforms part of a hybrid gene encoding additional polypeptide sequencesthat can be used, for example, in the production of a fusion protein.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Sambrook et al., 1989).Unless otherwise provided, ligation may be accomplished using knownbuffers and conditions with 10 units of T4 DNA ligase (“ligase”) per 0.5μg of approximately equimolar amounts of the DNA fragments to beligated.

As used herein, a “nucleic acid molecule” is comprised of at least onenucleotide base or one nucleotide base pair, depending on whether it issingle-stranded or double-stranded, respectively. Furthermore, a nucleicacid molecule may belong exclusively or chimerically to any group ofnucleotide-containing molecules, as exemplified by, but not limited to,the following groups of nucleic acid molecules: RNA, DNA, genomicnucleic acids, non-genomic nucleic acids, naturally occurring and notnaturally occurring nucleic acids, and synthetic nucleic acids. Thisincludes, by way of non-limiting example, nucleic acids associated withany organelle, such as the mitochondria, ribosomal RNA, and nucleic acidmolecules comprised chimerically of one or more components that are notnaturally occurring along with naturally occurring components.

Additionally, a “nucleic acid molecule” may contain in part one or morenon-nucleotide-based components as exemplified by, but not limited to,amino acids and sugars. Thus, by way of example, but not limitation, aribozyme that is in part nucleotide-based and in part protein-based isconsidered a “nucleic acid molecule”.

In addition, by way of example, but not limitation, a nucleic acidmolecule that is labeled with a detectable moiety, such as a radioactiveor alternatively a non-radioactive label, is likewise considered a“nucleic acid molecule”.

The terms “nucleic acid sequence coding for” or a “DNA coding sequenceof” or a “nucleotide sequence encoding” a particular enzyme—as well asother synonymous terms—refer to a DNA sequence which is transcribed andtranslated into an enzyme when placed under the control of appropriateregulatory sequences. A “promotor sequence” is a DNA regulatory regioncapable of binding RNA polymerase in a cell and initiating transcriptionof a downstream (3′ direction) coding sequence. The promoter is part ofthe DNA sequence. This sequence region has a start codon at its 3′terminus. The promoter sequence does include the minimum number of baseswhere elements necessary to initiate transcription at levels detectableabove background bind. However, after the RNA polymerase binds thesequence and transcription is initiated at the start codon (3′ terminuswith a promoter), transcription proceeds downstream in the 3′ direction.Within the promotor sequence will be found a transcription initiationsite (conveniently defined by mapping with nuclease S1) as well asprotein binding domains (consensus sequences) responsible for thebinding of RNA polymerase.

The terms “nucleic acid encoding an enzyme (protein)” or “DNA encodingan enzyme (protein)” or “polynucleotide encoding an enzyme (protein)”and other synonymous terms encompasses a polynucleotide which includesonly coding sequence for the enzyme as well as a polynucleotide whichincludes additional coding and/or non-coding sequence.

In one preferred embodiment, a “specific nucleic acid molecule species”is defined by its chemical structure, as exemplified by, but not limitedto, its primary sequence. In another preferred embodiment, a specific“nucleic acid molecule species” is defined by a function of the nucleicacid species or by a function of a product derived from the nucleic acidspecies. Thus, by way of non-limiting example, a “specific nucleic acidmolecule species” may be defined by one or more activities or propertiesattributable to it, including activities or properties attributable itsexpressed product.

The instant definition of “assembling a working nucleic acid sample intoa nucleic acid library” includes the process of incorporating a nucleicacid sample into a vector-based collection, such as by ligation into avector and transformation of a host. A description of relevant vectors,hosts, and other reagents as well as specific non-limiting examplesthereof are provided hereinafter. The instant definition of “assemblinga working nucleic acid sample into a nucleic acid library” also includesthe process of incorporating a nucleic acid sample into anon-vector-based collection, such as by ligation to adaptors. In oneaspect the adaptors can anneal to PCR primers to facilitateamplification by PCR.

Accordingly, in a non-limiting embodiment, a “nucleic acid library” iscomprised of a vector-based collection of one or more nucleic acidmolecules. In another preferred embodiment a “nucleic acid library” iscomprised of a non-vector-based collection of nucleic acid molecules. Inyet another preferred embodiment a “nucleic acid library” is comprisedof a combined collection of nucleic acid molecules that is in partvector-based and in part non-vector-based. In one aspect, the collectionof molecules comprising a library is searchable and separable accordingto individual nucleic acid molecule species.

The present invention provides a “nucleic acid construct” oralternatively a “nucleotide construct” or alternatively a “DNAconstruct”. The term “construct” is used herein to describe a molecule,such as a polynucleotide (e.g., a phytase polynucleotide) may optionallybe chemically bonded to one or more additional molecular moieties, suchas a vector, or parts of a vector. In a specific—but by no meanslimiting—aspect, a nucleotide construct is exemplified by a DNAexpression construct suitable for the transformation of a host cell.

“Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotides mayor may not have a 5′ phosphate. Those that do not will not ligate toanother oligonucleotide without adding a phosphate with an ATP in thepresence of a kinase. A synthetic oligonucleotide will ligate to afragment that has not been dephosphorylated.

A coding sequence is “operably linked to” another coding sequence whenRNA polymerase will transcribe the two coding sequences into a singlemRNA, which is then translated into a single polypeptide having aminoacids derived from both coding sequences. The coding sequences need notbe contiguous to one another so long as the expressed sequences areultimately processed to produce the desired protein.

The term “phytase-specific probe”, in the context of this method ofinvention, refers to probes that bind to nucleic acids encoding phytasepolypeptides, or to complementary sequences thereof, to a detectablygreater extent than to nucleic acids encoding other enzymes, or tocomplementary sequences thereof.

In a strict sense, the terms “phytate”, “phytic acid”, and “phytin”, maybe differentiated as folllows: “phytate” refers to an anionic form ofphytic acid; “phytic acid” refers to inositol hexaphosphate, a compoundthat occurs naturally in plants, including particularly plant leaves,and that may serve as a substrate for the enzyme phytase; and “phytin”refers to a salt of phytic acid, such as a calcium-magnesium salt ofphytic acid. It is understood, accordingly, that “phytate”, “phyticacid”, and “phytin” are chemically related and interconvertible formshaving a shared chemical structure. As used herein, therefore,“phytate”, “phytic acid”, and “phytin” are interchangeable terms in asmuch as they are highly related, similar, chemically interconvertible,and may all (either with or without the chemical interconversion) besubject to degredation by the novel phytase enzyme disclosed instantly.Accordingly, where only one of the terms “phytate”, “phytic acid”, or“phytin” is used in the descriptions of the methods disclosed herein, itis understood to function as a representative term that further refersto any substrate of the enzyme phytase including “phytase”, “phyticacid”, and “phytin”.

A “polynucleotide” is a molecule composed of 2 or more nucleotide basesor nucleotide base pairs.

A molecule having a “pre-form” or a “pro-form” refers to a molecule thatundergoes any combination of one or more covalent and noncovalentchemical modifications (e.g. glycosylation, proteolytic cleavage,dimerization or oligomerization, temperature-induced or pH-inducedconformational change, association with a co-factor, etc.) en route toattain a more mature molecular form having a property difference (e.g.an increase in activity) in comparison with the reference pro-formmolecule. When a precursor molecule in “pre-form” or in “pro-form” isable to undergo two or more chemical modification (e.g. two proteolyticcleavages, or a proteolytic cleavage and a change in glycosylation) enroute to the production of a mature molecule, the term “pre-pro-form”may also be used in reference to the precursor molecule. Accordingly, apre-pro-enzyme is an enzyme in “pre-pro-form”. Likewise, a pre-prohormone is a hormone in “pre-pro-form”.

As used herein, the term “reagent” includes phytase molecules of theinstant invention. In one aspect, such phytase molecules catalyze thehydrolysis of phytate to inositol and free phosphate with release ofminerals from the phytic acid complex. An exemplary phytase molecule isa phytase derived from Escherichia coli B. This exemplary enzyme isshown in FIGS. 1 a and 1 b, SEQ ID NO:2. Additionally, as used herein,the term “reagent” includes substrate reagents molecules of the instantinvention, such as phytate molecules. In one aspect, such phytatemolecules are found in foodstuffs, potential foodstuffs, byproducts offoodstuffs (both in vitro byproducts and in vivo byproducts, e.g. exvivo reaction products and animal excremental products), precursors offoodstuffs, and any other source of phytate.

“Recombinant” enzymes refer to enzymes produced by recombinant DNAtechniques, i.e., produced from cells transformed by an exogenous DNAconstruct encoding the desired enzyme. “Synthetic” enzymes are thoseprepared by chemical synthesis.

As known in the art “similarity” between two enzymes is determined bycomparing the amino acid sequence and its conserved amino acidsubstitutes of one enzyme to the sequence of a second enzyme. Similaritymay be determined by procedures which are well-known in the art, forexample, a BLAST program (Basic Local Alignment Search Tool at theNational Center for Biological Information).

The members of a pair of molecules (e.g., an antibody-antigen pair or anucleic acid pair) are said to “specifically bind” to each other if theybind to each other with greater affinity than to other, non-specificmolecules. For example, an antibody raised against an antigen to whichit binds more efficiently than to a non-specific protein can bedescribed as specifically binding to the antigen. (Similarly, a nucleicacid probe can be described as specifically binding to a nucleic acidtarget if it forms a specific duplex with the target by base pairinginteractions (see above).)

“Stringent hybridization conditions” means hybridization will occur onlyif there is at least 90% identity, or, at least 95% identity, or, atleast 97% identity between the sequences. See Sambrook et al., 1989.

Also included in the invention are polypeptides having sequences thatare “substantially identical” to the sequence of a phytase polypeptide,such as one of SEQ ID NO:1. A “substantially identical” amino acidsequence is a sequence that differs from a reference sequence orsequences only by conservative amino acid substitutions, for example,substitutions of one amino acid for another of the same class (e.g.,substitution of one hydrophobic amino acid, such as isoleucine, valine,leucine, or methionine, for another, or substitution of one polar aminoacid for another, such as substitution of arginine for lysine, glutamicacid for aspartic acid, or glutamine for asparagine).

Additionally a “substantially identical” amino acid sequence is asequence that differs from a reference sequence or sequences or by oneor more non-conservative substitutions, deletions, or insertions,particularly when such a substitution occurs at a site that is not theactive site the molecule, and provided that the polypeptide essentiallyretains its behavioural properties. For example, one or more amino acidscan be deleted from a phytase polypeptide, resulting in modification ofthe structure of the polypeptide, without significantly altering itsbiological activity. For example, amino- or carboxyl-terminal aminoacids that are not required for phytase biological activity can beremoved. Such modifications can result in the development of smalleractive phytase polypeptides.

The present invention provides a “substantially pure enzyme”. The term“substantially pure enzyme” is used herein to describe a molecule, suchas a polypeptide (e.g., a phytase polypeptide, or a fragment thereof)that is substantially free of other proteins, lipids, carbohydrates,nucleic acids, and other biological materials with which it is naturallyassociated. For example, a substantially pure molecule, such as apolypeptide, can be at least 60%, by dry weight, the molecule ofinterest. The purity of the polypeptides can be determined usingstandard methods including, e.g., polyacrylamide gel electrophoresis(e.g., SDS-PAGE), column chromatography (e.g., high performance liquidchromatography (HPLC)), and amino-terminal amino acid sequence analysis.

The present invention provides purified a recombinant enzyme thatcatalyzes the hydrolysis of phytate to inositol and free phosphate withrelease of minerals from the phytic acid complex. An exemplary purifiedenzyme is a phytase derived from Escherichia coli B. This exemplaryenzyme is shown in FIGS. 1 a and 1 b, SEQ ID NO:2.

The enzymes of the present invention include, in addition to an enzymeof FIGS. 1 a and 1 b (in particular the mature enzyme), polypeptideshaving sequences that are “substantially identical” to the sequence of aphytase polypeptide, such as one of SEQ ID 1.

In one embodiment, the phytase enzyme of SEQ ID NO:2 of the presentinvention has a molecular weight of about 47,056 kilodaltons as measuredby SDS-PAGE and inferred from the nucleotide sequence of the gene. ThepI is 6.70. The pH and temperature profile and stability data for thisenzyme is presented in FIG. 2. This purified enzyme may be used tocatalyze the hydrolysis of phytate to inositol and free phosphate wheredesired. The phytase enzyme of the present invention has a highthermostability; thus it is particularly serviceable for raisedtemperature and/or pressure applications including, but not limited to,the preparation of fish foodstuff pellets that will not dissolveprematurely in water.

The phytase polypeptide included in the invention can have the aminoacid sequences of the phytase shown in FIGS. 1 a and 1 b (SEQ ID NO:2).Phytase polypeptides, such as those isolated from E. coli B, can becharacterized by catalyzing the hydrolysis of phytate to inositol andfree phosphate with the release of minerals from the phytic acidcomplex.

Other phytase polypeptides used in the compositions (e.g., formulations,dietary supplements) and methods of the invention comprise phytases ofthe invention (e.g., a phytase having a sequence identity of at leastabout 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, ormore, or complete (100%) sequence identity (i.e., homology) to SEQ IDNO:2 (a phytase polypeptide); SEQ ID NO:10 (a phytase polypeptide); apolypeptide having sequence as set forth in SEQ ID NO:8 and having atleast one, or all, of the amino acid modifications W68E, Q84W, A95P,K97C, S168E, R181Y, N226C, Y277D, wherein the polypeptide has phytaseactivity) or other phytases, for example, the E. coli appA “wild type”phytase-encoding SEQ ID NO:7, or, a polypeptide sequence of SEQ ID NO:2or the E. coli appA “wild type” phytase SEQ ID NO:8. The length ofcomparison in determining amino acid sequence homology can be, forexample, at least 15 amino acids, and for example, at least 20, 25, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100 or more amino acids.

Homology or identity is often measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various deletions, substitutions andother modifications. The terms “homology” and “identity” in the contextof two or more nucleic acids or polypeptide sequences, refer to two ormore sequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same whencompared and aligned for maximum correspondence over a comparison windowor designated region as measured using any number of sequence comparisonalgorithms or by manual alignment and visual inspection.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared, however a database ofreference sequences can be used. When using a sequence comparisonalgorithm, test and reference sequences are entered into a computer,subsequence coordinates are designated, if necessary, and sequencealgorithm program parameters are designated. Default program parameterscan be used, or alternative parameters can be designated. The sequencecomparison algorithm then calculates the percent sequence identities forthe test sequences relative to the reference sequence, based on theprogram parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencefor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970,by the search for similarity method of person & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444, 1988, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection. Other algorithmsfor determining homology or identity include, for example, in additionto a BLAST program (Basic Local Alignment Search Tool at the NationalCenter for Biological Information), ALIGN, AMAS (Analysis of MultiplyAligned Sequences), AMPS (Protein Multiple Sequence Alignment), ASSET(Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN(Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProvedSearcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W,CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, LasVegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign,Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence AnalysisPackage), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC(Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP(Local Content Program), MACAW (Multiple Alignment Construction &Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN,PIMA (attem-Induced Multi-sequence Alignment), SAGA (Sequence Alignmentby Genetic Algorithm) and WHAT-IF. Such alignment programs can also beused to screen genome databases to identify polynucleotide sequenceshaving substantially identical sequences. A number of genome databasesare available, for example, a substantial portion of the human genome isavailable as part of the Human Genome Sequencing Project (J. Roach,http://weber.u.Washington.edu/˜roach/human_genome_progress 2.html)(Gibbs, 1995). At least twenty-one other genomes have already beensequenced, including, for example, M. genitalium (Fraser et al., 1995),M. jannaschii (Bult et al., 1996), H. influenzae (Fleischmann et al.,1995), E. coli (Blattner et al., 1997), and yeast (S. cerevisiae) (Meweset al., 1997), and D. melanogaster (Adams et al., 2000). Significantprogress has also been made in sequencing the genomes of model organism,such as mouse, C. elegans, and Arabadopsis sp. Several databasescontaining genomic information annotated with some functionalinformation are maintained by different organization, and are accessiblevia the internet.

One example of a useful algorithm is BLAST and BLAST 2.0 algorithms,which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402,1977, and Altschul et al., J. Mol. Biol. 215:403-410, 1990,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information.This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always>0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4 and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength of 3, and expectations (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915,1989) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and acomparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Natl. Acad. Sci. USA 90:5873, 1993). One measure of similarity providedby BLAST algorithm is the smallest sum probability (P(N)), whichprovides an indication of the probability by which a match between twonucleotide or amino acid sequences would occur by chance. For example, anucleic acid is considered similar to a references sequence if thesmallest sum probability in a comparison of the test nucleic acid to thereference nucleic acid is less than about 0.2, or, less than about 0.01,or, less than about 0.001.

In one embodiment, protein and nucleic acid sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”) Inparticular, five specific BLAST programs are used to perform thefollowing task:

-   -   (1) BLASTP and BLAST3 compare an amino acid query sequence        against a protein sequence database;    -   (2) BLASTN compares a nucleotide query sequence against a        nucleotide sequence database;    -   (3) BLASTX compares the six-frame conceptual translation        products of a query nucleotide sequence (both strands) against a        protein sequence database;    -   (4) TBLASTN compares a query protein sequence against a        nucleotide sequence database translated in all six reading        frames (both strands); and    -   (5) TBLASTX compares the six-frame translations of a nucleotide        query sequence against the six-frame translations of a        nucleotide sequence database.

The BLAST programs identify homologous sequences by identifying similarsegments, which are referred to herein as “high-scoring segment pairs,”between a query amino or nucleic acid sequence and a test sequence whichis can be obtained from a protein or nucleic acid sequence database.High-scoring segment pairs can be identified (ie., aligned) by means ofa scoring matrix, many of which are known in the art. In one aspect, thescoring matrix used is the BLOSUM62 matrix (Gonnet et al., Science256:1443-1445, 1992; Henikoff and Henikoff, Proteins 17:49-61, 1993).The PAM or PAM250 matrices may also be used (see, e.g., Schwartz andDayhoff, eds., 1978, Matrices for Detecting Distance Relationships:Atlas of Protein Sequence and Structure, Washington: National BiomedicalResearch Foundation). BLAST programs are accessible through the U.S.National Library of Medicine.

The parameters used with the above algorithms may be adapted dependingon the sequence length and degree of homology studied. In someembodiments, the parameters may be the default parameters used by thealgorithms in the absence of instructions from the user.

The present invention further relates to an enzyme which has the deducedamino acid sequence of FIGS. 1 a and 1 b, as well as analogs,derivatives, and fragments of such enzyme.

An analog, derivative, or fragment of the enzyme of FIGS. 1 a and 1 bmay be (a) one in which one or more of the amino acid residues aresubstituted with an amino acid residue which is not encoded by thegenetic code, or (b) one in which one or more of the amino acid residuesincludes a substituent group, or (c) one in which the mature enzyme isfused with another compound, such as a compound to increase thehalf-life of the enzyme (for example, polyethylene glycol), or (d) toprovide a label or a tag, such as a 6×His tag or a green fluorescentprotein tag, (e) one in which the additional amino acids are fused tothe mature enzyme, such as a leader or secretory sequence or a sequencewhich is employed for purification of the mature enzyme or a proproteinsequence. Such analogs, derivatives, and fragments are deemed to bewithin the scope of those skilled in the art from the teachings herein.

A variant, e.g., a “fragment”, “analog” or “derivative” enzyme, andreference enzyme may differ in amino acid sequence by one or moresubstitutions, additions, deletions, fusions and truncations, which maybe present in any combination.

Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

Thus, in a particular non-limiting exemplification, a substitution canbe comprised of a substitution of one amino acid by another amino acidwith a like property. In another particular non-limitingexemplification, a substitution can be comprised of a substitution of anamino acid by an unlike amino acid, where the change is non-inhibitoryor silent or improved with respect to at least one enzyme property.

Additionally, in a non-limiting exemplification, an addition can becomprised of an addition either at the amino or the carboxy terminal ofthe protein or alternatively between the terminal sites, where thechange is change is non-inhibitory or silent or improved with respect toat least one enzyme property.

In another particular non-limiting exemplification, a change can becomprised of a plurality of modifications, including substitutions,additions, deletions, fusions and/or truncations, in the enzyme encodedby the reference polynucleotide (SEQ ID NO:1, such that, irrespective ofthe effects of the individual modifications, when taken together as aset, the effect of the modifications is non-inhibitory or silent orimproved with respect to at least one enzyme property.

In one aspect, a phytase variant retains substantially the samebiological function and activity as the reference polypeptide from whichit varies.

The term “variant” refers to polynucleotides or polypeptides of theinvention modified at one or more base pairs, codons, introns, exons, oramino acid residues (respectively) yet still retain the biologicalactivity of a phytase of the invention. Variants can be produced by anynumber of means including methods such as, for example, error-prone PCR,shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexualPCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, ligation reassembly, Gene Site Saturation Mutagenesis™(GSSM™) and any combination thereof as discussed more fully below. Theinvention also provides methods for modifying any phytase using theseexemplary or other methods.

In accordance with an aspect of the present invention, there areprovided isolated nucleic acid molecules (polynucleotides) which encodefor the mature enzyme having the deduced amino acid sequence of FIGS. 1a and 1 b.

The polynucleotide encoding SEQ ID NO:2 was originally isolated fromgenomic DNA recovered from Escherichia coli B as described below. Itcontains an open reading frame encoding a protein of 432 amino acidresidues.

In accordance with another aspect of the present invention, there isprovided an isolated polynucleotide encoding an exemplary enzyme of thepresent invention (SEQ ID NO:1) comprising the DNA of FIGS. 1 a and 1 b.

The present invention also relates to polynucleotides which differ fromthe reference polynucleotide such that the changes are silent changes,for example the changes do not alter the amino acid sequence encoded bythe polynucleotide. The present invention also relates to nucleotidechanges which result in amino acid substitutions, additions, deletions,fusions and truncations in the enzyme encoded by the referencepolynucleotide (SEQ ID NO:1). In a preferred aspect of the inventionthese enzymes retain about the same biological action as the enzymeencoded by the reference polynucleotide.

The invention also provides isolated nucleic acid molecules that encodethe phytase polypeptide described above. For example, nucleic acids thatencode SEQ ID NO:2 are included in the invention. These nucleic acidscan contain naturally occurring nucleotide sequences, or sequences thatdiffer from those of the naturally occurring nucleic acids that encodephytases, but encode the same amino acids, due to the degeneracy of thegenetic code. The nucleic acids of the invention can contain DNA or RNAnucleotides, or combinations or modifications thereof. Exemplary nucleicacids of the invention are shown in SEQ ID NO:1.

The polynucleotide of the present invention may be in the form of DNAwhich DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may bedouble-stranded or single-stranded, and if single stranded may be thecoding strand or non-coding (anti-sense) strand. The coding sequencewhich encodes the mature enzyme may be identical to the coding sequencesshown in FIGS. 1 a and 1 b and/or that of the deposited clone (SEQ IDNO:1), or may be a different coding sequence which coding sequence, as aresult of the redundancy or degeneracy of the genetic code, encodes thesame mature enzyme as the DNA of FIGS. 1 a and 1 b (e.g., SEQ ID NO:1).

The polynucleotide which encodes for the mature enzyme of FIGS. 1 a and1 b (e.g., SEQ ID NO:2) may include, but is not limited to: only thecoding sequence for the mature enzyme; the coding sequence for themature enzyme and additional coding sequence such as a leader sequenceor a proprotein sequence; the coding sequence for the mature enzyme (andoptionally additional coding sequence) and non-coding sequence, such asintrons or non-coding sequence 5′ and/or 3′ of the coding sequence forthe mature enzyme.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the enzyme having the deduced amino acid sequence ofFIGS. 1 a and 1 b (e.g., SEQ ID NO:2). The variant of the polynucleotidemay be a naturally occurring allelic variant of the polynucleotide or anon-naturally occurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature enzyme as shown in FIGS. 1 a and 1 b as well as variants of suchpolynucleotides which variants encode for a fragment, derivative oranalog of the enzyme of FIGS. 1 a and 1 b. Such nucleotide variantsinclude deletion variants, substitution variants and addition orinsertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIGS. 1 a and 1 b. As known in the art, an allelic variant isan alternate form of a polynucleotide sequence which may have asubstitution, deletion or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded enzyme.

Phytase variants, including variant of the phytases of the invention,the phytases described herein, or, any phytase, can be produced by anynumber of means including methods such as, for example, error-prone PCR,shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexualPCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, ligation reassembly, GSSM™ and any combination thereof.

In one aspect, a non-stochastic method termed synthetic ligationreassembly (SLR), that is somewhat related to stochastic shuffling, savethat the nucleic acid building blocks are not shuffled or concatenatedor chimerized randomly, but rather are assembled non-stochastically canbe used to create variants.

The SLR method does not depend on the presence of a high level ofhomology between polynucleotides to be shuffled. The invention can beused to non-stochastically generate libraries (or sets) of progenymolecules comprised of over 10¹⁰⁰ different chimeras. Conceivably, SLRcan even be used to generate libraries comprised of over 10¹⁰⁰⁰different progeny chimeras.

Thus, in one aspect, the invention provides a non-stochastic method ofproducing a set of finalized chimeric nucleic acid molecules having anoverall assembly order that is chosen by design, which method iscomprised of the steps of generating by design a plurality of specificnucleic acid building blocks having serviceable mutually compatibleligatable ends, and assembling these nucleic acid building blocks, suchthat a designed overall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus, in one aspect, the overall assembly order inwhich the nucleic acid building blocks can be coupled is specified bythe design of the ligatable ends and, if more than one assembly step isto be used, then the overall assembly order in which the nucleic acidbuilding blocks can be coupled is also specified by the sequential orderof the assembly step(s). In a one embodiment of the invention, theannealed building pieces are treated with an enzyme, such as a ligase(e.g., T4 DNA ligase) to achieve covalent bonding of the buildingpieces.

In a another embodiment, the design of nucleic acid building blocks isobtained upon analysis of the sequences of a set of progenitor nucleicacid templates that serve as a basis for producing a progeny set offinalized chimeric nucleic acid molecules. These progenitor nucleic acidtemplates thus serve as a source of sequence information that aids inthe design of the nucleic acid building blocks that are to bemutagenized, i.e. chimerized or shuffled.

In one exemplification, the invention provides for the chimerization ofa family of related genes and their encoded family of related products.In a particular exemplification, the encoded products are enzymes.Enzymes and polypeptides of the invention can be mutagenized inaccordance with the methods described herein.

Thus according to one aspect of the invention, the sequences of aplurality of progenitor nucleic acid templates are aligned in order toselect one or more demarcation points, which demarcation points can belocated at an area of homology. The demarcation points can be used todelineate the boundaries of nucleic acid building blocks to begenerated. Thus, the demarcation points identified and selected in theprogenitor molecules serve as potential chimerization points in theassembly of the progeny molecules.

Typically a serviceable demarcation point is an area of homology(comprised of at least one homologous nucleotide base) shared by atleast two progenitor templates, but the demarcation point can be an areaof homology that is shared by at least half of the progenitor templates,at least two thirds of the progenitor templates, at least three fourthsof the progenitor templates, and can be at almost all of the progenitortemplates. A serviceable demarcation point can be an area of homologythat is shared by all of the progenitor templates.

In a one embodiment, the ligation reassembly process is performedexhaustively in order to generate an exhaustive library. In other words,all possible ordered combinations of the nucleic acid building blocksare represented in the set of finalized chimeric nucleic acid molecules.At the same time, the assembly order (i.e. the order of assembly of eachbuilding block in the 5′ to 3 sequence of each finalized chimericnucleic acid) in each combination is by design (or non-stochastic).Because of the non-stochastic nature of the method, the possibility ofunwanted side products is greatly reduced.

In another embodiment, the method provides that, the ligation reassemblyprocess is performed systematically, for example in order to generate asystematically compartmentalized library, with compartments that can bescreened systematically, e.g., one by one. In other words the inventionprovides that, through the selective and judicious use of specificnucleic acid building blocks, coupled with the selective and judicioususe of sequentially stepped assembly reactions, an experimental designcan be achieved where specific sets of progeny products are made in eachof several reaction vessels. This allows a systematic examination andscreening procedure to be performed. Thus, it allows a potentially verylarge number of progeny molecules to be examined systematically insmaller groups.

Because of its ability to perform chimerizations in a manner that ishighly flexible yet exhaustive and systematic as well, particularly whenthere is a low level of homology among the progenitor molecules, theinstant invention provides for the generation of a library (or set)comprised of a large number of progeny molecules. Because of thenon-stochastic nature of the instant ligation reassembly invention, theprogeny molecules generated can comprise a library of finalized chimericnucleic acid molecules having an overall assembly order that is chosenby design. In a particularly embodiment, such a generated library iscomprised of greater than 10³ to greater than 10¹⁰⁰⁰ different progenymolecular species.

In one aspect, a set of finalized chimeric nucleic acid molecules,produced as described is comprised of a polynucleotide encoding apolypeptide. According to one embodiment, this polynucleotide is a gene,which may be a man-made gene. According to another embodiment, thispolynucleotide is a gene pathway, which may be a man-made gene pathway.The invention provides that one or more man-made genes generated by theinvention may be incorporated into a man-made gene pathway, such aspathway operable in a eukaryotic organism (including a plant).

In another exemplifaction, the synthetic nature of the step in which thebuilding blocks are generated allows the design and introduction ofnucleotides (e.g., one or more nucleotides, which may be, for example,codons or introns or regulatory sequences) that can later be optionallyremoved in an in vitro process (e.g., by mutageneis) or in an in vivoprocess (e.g., by utilizing the gene splicing ability of a hostorganism). It is appreciated that in many instances the introduction ofthese nucleotides may also be desirable for many other reasons inaddition to the potential benefit of creating a serviceable demarcationpoint.

Thus, according to another embodiment, the invention provides that anucleic acid building block can be used to introduce an intron. Thus,the invention provides that functional introns may be introduced into aman-made gene of the invention. The invention also provides thatfunctional introns may be introduced into a man-made gene pathway of theinvention. Accordingly, the invention provides for the generation of achimeric polynucleotide that is a man-made gene containing one (or more)artificially introduced intron(s).

Accordingly, the invention also provides for the generation of achimeric polynucleotide that is a man-made gene pathway containing one(or more) artificially introduced intron(s). In one aspect, theartificially introduced intron(s) are functional in one or more hostcells for gene splicing much in the way that naturally-occurring intronsserve functionally in gene splicing. The invention provides a process ofproducing man-made intron-containing polynucleotides to be introducedinto host organisms for recombination and/or splicing.

A man-made gene produced using the invention can also serve as asubstrate for recombination with another nucleic acid. Likewise, aman-made gene pathway produced using the invention can also serve as asubstrate for recombination with another nucleic acid. In a preferredinstance, the recombination is facilitated by, or occurs at, areas ofhomology between the man-made intron-containing gene and a nucleic acidwith serves as a recombination partner. In a particularly preferredinstance, the recombination partner may also be a nucleic acid generatedby the invention, including a man-made gene or a man-made gene pathway.Recombination may be facilitated by or may occur at areas of homologythat exist at the one (or more) artificially introduced intron(s) in theman-made gene.

The synthetic ligation reassembly method of the invention utilizes aplurality of nucleic acid building blocks, each of which can have twoligatable ends. The two ligatable ends on each nucleic acid buildingblock may be two blunt ends (i.e. each having an overhang of zeronucleotides), or can be one blunt end and one overhang, or, twooverhangs.

A serviceable overhang for this purpose may be a 3′ overhang or a 5′overhang. Thus, a nucleic acid building block may have a 3′ overhang oralternatively a 5′ overhang or alternatively two 3′ overhangs oralternatively two 5′ overhangs. The overall order in which the nucleicacid building blocks are assembled to form a finalized chimeric nucleicacid molecule is determined by purposeful experimental design and is notrandom.

A nucleic acid building block can be generated by chemical synthesis oftwo single-stranded nucleic acids (also referred to as single-strandedoligos) and contacting them so as to allow them to anneal to form adouble-stranded nucleic acid building block.

A double-stranded nucleic acid building block can be of variable size.The sizes of these building blocks can be small or large. Preferredsizes for building block range from 1 base pair (not including anyoverhangs) to 100,000 base pairs (not including any overhangs). Otherpreferred size ranges are also provided, which have lower limits of from1 bp to 10,000 bp (including every integer value in between), and upperlimits of from 2 bp to 100,000 bp (including every integer value inbetween).

Many methods exist by which a double-stranded nucleic acid buildingblock can be generated that is serviceable for the invention; and theseare known in the art and can be readily performed by the skilledartisan.

According to one embodiment, a double-stranded nucleic acid buildingblock is generated by first generating two single stranded nucleic acidsand allowing them to anneal to form a double-stranded nucleic acidbuilding block. The two strands of a double-stranded nucleic acidbuilding block may be complementary at every nucleotide apart from anythat form an overhang; thus containing no mismatches, apart from anyoverhang(s). According to another embodiment, the two strands of adouble-stranded nucleic acid building block are complementary at fewerthan every nucleotide apart from any that form an overhang. Thus,according to this embodiment, a double-stranded nucleic acid buildingblock can be used to introduce codon degeneracy. In one aspect the codondegeneracy is introduced using the site-saturation mutagenesis describedherein, using one or more N,N,G/T cassettes or alternatively using oneor more N,N,N cassettes.

The in vivo recombination method of the invention can be performedblindly on a pool of unknown hybrids or alleles of a specificpolynucleotide or sequence. However, it is not necessary to know theactual DNA or RNA sequence of the specific polynucleotide.

The approach of using recombination within a mixed population of genescan be useful for the generation of any useful proteins, for example,interleukin I, antibodies, tPA and growth hormone. This approach may beused to generate proteins having altered specificity or activity. Theapproach may also be useful for the generation of hybrid nucleic acidsequences, for example, promoter regions, introns, exons, enhancersequences, 31 untranslated regions or 51 untranslated regions of genes.Thus this approach may be used to generate genes having increased ratesof expression. This approach may also be useful in the study ofrepetitive DNA sequences. Finally, this approach may be useful to mutateribozymes or aptamers.

In one aspect variants of the polynucleotides and polypeptides describedherein are obtained by the use of repeated cycles of reductivereassortment, recombination and selection which allow for the directedmolecular evolution of highly complex linear sequences, such as DNA, RNAor proteins thorough recombination.

In vivo shuffling of molecules is useful in providing variants and canbe performed utilizing the natural property of cells to recombinemultimers. While recombination in vivo has provided the major naturalroute to molecular diversity, genetic recombination remains a relativelycomplex process that involves 1) the recognition of homologies; 2)strand cleavage, strand invasion, and metabolic steps leading to theproduction of recombinant chiasma; and finally 3) the resolution ofchiasma into discrete recombined molecules. The formation of the chiasmarequires the recognition of homologous sequences.

In another embodiment, the invention includes a method for producing ahybrid polynucleotide from at least a first polynucleotide and a secondpolynucleotide. The invention can be used to produce a hybridpolynucleotide by introducing at least a first polynucleotide and asecond polynucleotide which share at least one region of partialsequence homology into a suitable host cell. The regions of partialsequence homology promote processes which result in sequencereorganization producing a hybrid polynucleotide. The term “hybridpolynucleotide”, as used herein, is any nucleotide sequence whichresults from the method of the present invention and contains sequencefrom at least two original polynucleotide sequences. Such hybridpolynucleotides can result from intermolecular recombination eventswhich promote sequence integration between DNA molecules. In addition,such hybrid polynucleotides can result from intramolecular reductivereassortment processes which utilize repeated sequences to alter anucleotide sequence within a DNA molecule.

The invention provides a means for generating hybrid polynucleotideswhich may encode biologically active hybrid polypeptides (e.g., a hybridphytase). In one aspect, the original polynucleotides encodebiologically active polypeptides. The method of the invention producesnew hybrid polypeptides by utilizing cellular processes which integratethe sequence of the original polynucleotides such that the resultinghybrid polynucleotide encodes a polypeptide demonstrating activitiesderived from the original biologically active polypeptides. For example,the original polynucleotides may encode a particular enzyme fromdifferent microorganisms. An enzyme encoded by a first polynucleotidefrom one organism or variant may, for example, function effectivelyunder a particular environmental condition, e.g., high salinity. Anenzyme encoded by a second polynucleotide from a different organism orvariant may function effectively under a different environmentalcondition, such as extremely high temperatures. A hybrid polynucleotidecontaining sequences from the first and second original polynucleotidesmay encode an enzyme which exhibits characteristics of both enzymesencoded by the original polynucleotides. Thus, the enzyme encoded by thehybrid polynucleotide may function effectively under environmentalconditions shared by each of the enzymes encoded by the first and secondpolynucleotides, e.g., high salinity and extreme temperatures.

Enzymes encoded by original polynucleotides include, but are not limitedto, phytases. A hybrid polypeptide resulting from the method of theinvention may exhibit specialized enzyme activity not displayed in theoriginal enzymes. For example, following recombination and/or reductivereassortment of polynucleotides encoding hydrolase activities, theresulting hybrid polypeptide encoded by a hybrid polynucleotide can bescreened for specialized hydrolase activities obtained from each of theoriginal enzymes, i.e., the type of bond on which the hydrolase acts andthe temperature at which the hydrolase functions. Thus, for example, thehydrolase may be screened to ascertain those chemical functionalitieswhich distinguish the hybrid hydrolase from the original hydrolyases,such as: (a) amide (peptide bonds), i.e., proteases; (b) ester bonds,i.e., esterases and lipases; (c) acetals, i.e., glycosidases and, forexample, the temperature, pH or salt concentration at which the hybridpolypeptide functions.

Sources of the original polynucleotides may be isolated from individualorganisms (“isolates”), collections of organisms that have been grown indefined media (“enrichment cultures”), or, uncultivated organisms(“environmental samples”). The use of a culture-independent approach toderive polynucleotides encoding novel bioactivities from environmentalsamples is most preferable since it allows one to access untappedresources of biodiversity.

“Environmental libraries” are generated from environmental samples andrepresent the collective genomes of naturally occurring organismsarchived in cloning vectors that can be propagated in suitableprokaryotic hosts. Because the cloned DNA is initially extracteddirectly from environmental samples, the libraries are not limited tothe small fraction of prokaryotes that can be grown in pure culture.Additionally, a normalization of the environmental DNA present in thesesamples could allow more equal representation of the DNA from all of thespecies present in the original sample. This can dramatically increasethe efficiency of finding interesting genes from minor constituents ofthe sample which may be under-represented by several orders of magnitudecompared to the dominant species.

For example, gene libraries generated from one or more uncultivatedmicroorganisms are screened for an activity of interest. Potentialpathways encoding bioactive molecules of interest are first captured inprokaryotic cells in the form of gene expression libraries.Polynucleotides encoding activities of interest are isolated from suchlibraries and introduced into a host cell. The host cell is grown underconditions which promote recombination and/or reductive reassortmentcreating potentially active biomolecules with novel or enhancedactivities.

The microorganisms from which the polynucleotide may be prepared includeprokaryotic microorganisms, such as Xanthobacter, Eubacteria andArchaebacteria, and lower eukaryotic microorganisms such as fungi, somealgae and protozoa. Polynucleotides may be isolated from environmentalsamples in which case the nucleic acid may be recovered withoutculturing of an organism or recovered from one or more culturedorganisms. In one aspect, such microorganisms may be extremophiles, suchas hyperthermophiles, psychrophiles, psychrotrophs, halophiles,barophiles and acidophiles. Polynucleotides encoding enzymes isolatedfrom extremophilic microorganisms are particularly preferred. Suchenzymes may function at temperatures above 100° C. in terrestrial hotsprings and deep sea thermal vents, at temperatures below 0° C. inarctic waters, in the saturated salt environment of the Dead Sea, at pHvalues around 0 in coal deposits and geothermal sulfur-rich springs, orat pH values greater than 11 in sewage sludge. For example, severalesterases and lipases cloned and expressed from extremophilic organismsshow high activity throughout a wide range of temperatures and pHs.

Polynucleotides selected and isolated as hereinabove described areintroduced into a suitable host cell. A suitable host cell is any cellwhich is capable of promoting recombination and/or reductivereassortment. The selected polynucleotides can be already in a vectorwhich includes appropriate control sequences. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or, the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, or electroporation (Davis et al., 1986).

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as Bacillus (e.g., Bacillus cereus), E. coli,Streptomyces, Salmonella typhimurium; fungal cells, yeast; insect cellssuch as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COSor Bowes melanoma; adenoviruses; and plant cells. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

With particular references to various mammalian cell culture systemsthat can be employed to express recombinant protein, examples ofmammalian expression systems include the COS-7 lines of monkey kidneyfibroblasts, described in “SV40-transformed simian cells support thereplication of early SV40 mutants” (Gluzman, 1981), and other cell linescapable of expressing a compatible vector, for example, the C127, 3T3,CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprisean origin of replication, a suitable promoter and enhancer, and also anynecessary ribosome binding sites, polyadenylation site, splice donor andacceptor sites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

Host cells containing the polynucleotides of interest can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying genes. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothe ordinarily skilled artisan. The clones which are identified ashaving the specified enzyme activity may then be sequenced to identifythe polynucleotide sequence encoding an enzyme having the enhancedactivity.

In another aspect, methods can be used to generate novel polynucleotidesencoding biochemical pathways from one or more operons or gene clustersor portions thereof. For example, bacteria and many eukaryotes have acoordinated mechanism for regulating genes whose products are involvedin related processes. The genes are clustered, in structures referred toas “gene clusters,” on a single chromosome or immediately adjacent toone another and are transcribed together under the control of a singleregulatory sequence, including a single promoter which initiatestranscription of the entire cluster. Thus, a gene cluster is a group ofadjacent genes that are either identical or related, usually as to theirfunction. An example of a biochemical pathway encoded by gene clustersis a polyketide pathway. Polyketides are molecules which are anextremely rich source of bioactivities, including antibiotics (such astetracyclines and erythromycin), anti-cancer agents (daunomycin),immunosuppressants (FK506 and rapamycin), and veterinary products(monensin). Many polyketides (produced by polyketide synthases) arevaluable as therapeutic agents. Polyketide synthases are multifunctionalenzymes that catalyze the biosynthesis of an enormous variety of carbonchains differing in length and patterns of functionality andcyclization. Polyketide synthase genes fall into gene clusters and atleast one type (designated type I) of polyketide synthases have largesize genes and enzymes, complicating genetic manipulation and in vitrostudies of these genes/proteins.

Gene cluster DNA can be isolated from different organisms and ligatedinto vectors, particularly vectors containing expression regulatorysequences which can control and regulate the production of a detectableprotein or protein-related array activity from the ligated geneclusters. Use of vectors which have an exceptionally large capacity forexogenous DNA introduction are particularly appropriate for use withsuch gene clusters and are described by way of example herein to includethe f-factor (or fertility factor) of E. coli. This f-factor of E coliis a plasmid which affects high-frequency transfer of itself duringconjugation and is ideal to achieve and stably propagate large DNAfragments, such as gene clusters from mixed microbial samples. Onceligated into an appropriate vector, two or more vectors containingdifferent phytase gene clusters can be introduced into a suitable hostcell. Regions of partial sequence homology shared by the gene clusterswill promote processes which result in sequence reorganization resultingin a hybrid gene cluster. The novel hybrid gene cluster can then bescreened for enhanced activities not found in the original geneclusters.

Therefore, in a one embodiment, the invention relates to a method forproducing a biologically active hybrid polypeptide and screening such apolypeptide for enhanced activity by:

-   -   1) introducing at least a first polynucleotide in operable        linkage and a second polynucleotide in operable linkage, said at        least first polynucleotide and second polynucleotide sharing at        least one region of partial sequence homology, into a suitable        host cell;    -   2) growing the host cell under conditions which promote sequence        reorganization resulting in a hybrid polynucleotide in operable        linkage;    -   3) expressing a hybrid polypeptide encoded by the hybrid        polynucleotide;    -   4) screening the hybrid polypeptide under conditions which        promote identification of enhanced biological activity; and    -   5) isolating the a polynucleotide encoding the hybrid        polypeptide.

Methods for screening for various enzyme activities are known to thoseof skill in the art and are discussed throughout the presentspecification. Such methods may be employed when isolating thepolypeptides and polynucleotides of the invention.

As representative examples of expression vectors which may be used theremay be mentioned viral particles, baculovirus, bacteriophage insertionvectors or replacement vectors, phage, plasmids, phagemids, cosmids,fosmids, bacterial artificial chromosomes (BAC), viral DNA (e.g.,vaccinia, adenovirus, foul pox virus, pseudorabies and derivatives ofSV40), P1-based artificial chromosomes (PAC), yeast plasmids, yeastartificial chromosomes (YAC), and any other vectors specific forspecific hosts of interest (such as Bacillus, Aspergillus and yeast).Thus, for example, the DNA may be included in any one of a variety ofexpression vectors for expressing a polypeptide. Such vectors includechromosomal, nonchromosomal and synthetic DNA sequences. Large numbersof suitable vectors are known to those of skill in the art, and arecommercially available. The following vectors are provided by way ofexample; Bacterial: pQE vectors (Qiagen), pBluescript® plasmids, pNHvectors, (lambda-ZAP® vectors (Stratagene); ptrc99a, pKK223-3, pDR540,pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5 (Stratagene), pSVK3, pBPV,pMSG, pSVLSV40 (Pharmacia>. However, any other plasmid or other vectormay be used so long as they are replicable and viable in the host. Lowcopy number or high copy number vectors may be employed with the presentinvention.

An exemplary vector for use in the present invention contains anf-factor origin replication. The f-factor (or fertility factor) in E.coli is a plasmid which effects high frequency transfer of itself duringconjugation and less frequent transfer of the bacterial chromosomeitself. A particularly preferred embodiment is to use cloning vectors,referred to as “fosmids” or bacterial artificial chromosome (BAC)vectors. These are derived from E. coli f-factor which is able to stablyintegrate large segments of genomic DNA. When integrated with DNA from amixed uncultured environmental sample, this makes it possible to achievelarge genomic fragments in the form of a stable “environmental DNAlibrary.”

An exemplary vector for use in the present invention is a cosmid vector.Cosmid vectors were originally designed to clone and propagate largesegments of genomic DNA. Cloning into cosmid vectors is described indetail in “Molecular Cloning: A laboratory Manual” (Sambrook et al.,1989).

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct RNAsynthesis. Particular named bacterial promoters include lacI, lacZ, T3,T7, gpt, lambda P_(R), P_(L) and trp. Eukaryotic promoters include CMVimmediate early, HSV thymidine kinase, early and late SV40, LTRs fromretrovirus, and mouse metallothionein-I. Selection of the appropriatevector and promoter is well within the level of ordinary skill in theart. The expression vector also contains a ribosome binding site fortranslation initiation and a transcription terminator. The vector mayalso include appropriate sequences for amplifying expression. Promoterregions can be selected from any desired gene using CAT (chloranphenicoltransferase) vectors or other vectors with selectable markers. Inaddition, the expression vectors can contain one or more selectablemarker genes to provide a phenotypic trait for selection of transformedhost cells such as dihydrofolate reductase or neomycin resistance foreukaryotic cell culture, or tetracycline or ampicillin resistance in E.coli.

In vivo reassortment is focused on “inter-molecular” processescollectively referred to as “recombination” which in bacteria, isgenerally viewed as a “RecA-dependent” phenomenon. The invention canrely on recombination processes of a host cell to recombine andre-assort sequences, or the cells' ability to mediate reductiveprocesses to decrease the complexity of quasi-repeated sequences in thecell by deletion. This process of “reductive reassortment” occurs by an“intra-molecular”, RecA-independent process.

Therefore, in another aspect of the invention, variant polynucleotidescan be generated by the process of reductive reassortment. The methodinvolves the generation of constructs containing consecutive sequences(original encoding sequences), their insertion into an appropriatevector, and their subsequent introduction into an appropriate host cell.The reassortment of the individual molecular identities occurs bycombinatorial processes between the consecutive sequences in theconstruct possessing regions of homology, or between quasi-repeatedunits. The reassortment process recombines and/or reduces the complexityand extent of the repeated sequences, and results in the production ofnovel molecular species. Various treatments may be applied to enhancethe rate of reassortment. These could include treatment withultra-violet light, or DNA damaging chemicals, and/or the use of hostcell lines displaying enhanced levels of “genetic instability”. Thus thereassortment process may involve homologous recombination or the naturalproperty of quasi-repeated sequences to direct their own evolution.

Repeated or “quasi-repeated” sequences play a role in geneticinstability. In the present invention, “quasi-repeats” are repeats thatare not restricted to their original unit structure. Quasi-repeatedunits can be presented as an array of sequences in a construct;consecutive units of similar sequences. Once ligated, the junctionsbetween the consecutive sequences become essentially invisible and thequasi-repetitive nature of the resulting construct is now continuous atthe molecular level. The deletion process the cell performs to reducethe complexity of the resulting construct operates between thequasi-repeated sequences. The quasi-repeated units provide a practicallylimitless repertoire of templates upon which slippage events can occur.The constructs containing the quasi-repeats thus effectively providesufficient molecular elasticity that deletion (and potentiallyinsertion) events can occur virtually anywhere within thequasi-repetitive units.

When the quasi-repeated sequences are all ligated in the sameorientation, for instance head to tail or vice versa, the cell cannotdistinguish individual units. Consequently, the reductive process canoccur throughout the sequences. In contrast, when for example, the unitsare presented head to head, rather than head to tail, the inversiondelineates the endpoints of the adjacent unit so that deletion formationwill favor the loss of discrete units. Thus, it is preferable with thepresent method that the sequences are in the same orientation. Randomorientation of quasi-repeated sequences will result in the loss ofreassortment efficiency, while consistent orientation of the sequenceswill offer the highest efficiency. However, while having fewer of thecontiguous sequences in the same orientation decreases the efficiency,it may still provide sufficient elasticity for the effective recovery ofnovel molecules. Constructs can be made with the quasi-repeatedsequences in the same orientation to allow higher efficiency.

Sequences can be assembled in a head to tail orientation using any of avariety of methods, including the following:

-   -   a) Primers that include a poly-A head and poly-T tail which when        made single-stranded would provide orientation can be utilized.        This is accomplished by having the first few bases of the        primers made from RNA and hence easily removed RNAseH.    -   b) Primers that include unique restriction cleavage sites can be        utilized. Multiple sites, a battery of unique sequences, and        repeated synthesis and ligation steps would be required.    -   c) The inner few bases of the primer could be thiolated and an        exonuclease used to produce properly tailed molecules.

The recovery of the re-assorted sequences relies on the identificationof cloning vectors with a reduced RI. The re-assorted encoding sequencescan then be recovered by amplification. The products are re-cloned andexpressed. The recovery of cloning vectors with reduced RI can beeffected by:

-   1) The use of vectors only stably maintained when the construct is    reduced in complexity.-   2) The physical recovery of shortened vectors by physical    procedures. In this case, the cloning vector would be recovered    using standard plasmid isolation procedures and size fractionated on    either an agarose gel, or column with a low molecular weight cut off    utilizing standard procedures.-   3) The recovery of vectors containing interrupted genes which can be    selected when insert size decreases.-   4) The use of direct selection techniques with an expression vector    and the appropriate selection.

Encoding sequences (for example, genes) from related organisms maydemonstrate a high degree of homology and encode quite diverse proteinproducts. These types of sequences are particularly useful in thepresent invention as quasi-repeats. However, while the examplesillustrated below demonstrate the reassortment of nearly identicaloriginal encoding sequences (quasi-repeats), this process is not limitedto such nearly identical repeats.

The following example demonstrates a method of the invention. Encodingnucleic acid sequences (quasi-repeats) derived from three (3) uniquespecies are depicted. Each sequence encodes a protein with a distinctset of properties. Each of the sequences differs by a single or a fewbase pairs at a unique position in the sequence which are designated“A”, “B” and “C”. The quasi-repeated sequences are separately orcollectively amplified and ligated into random assemblies such that allpossible permutations and combinations are available in the populationof ligated molecules. The number of quasi-repeat units can be controlledby the assembly conditions. The average number of quasi-repeated unitsin a construct is defined as the repetitive index (RI).

Once formed, the constructs may, or may not be size fractionated on anagarose gel according to published protocols, inserted into a cloningvector, and transfected into an appropriate host cell. The cells arethen propagated and “reductive reassortment” is effected. The rate ofthe reductive reassortment process may be stimulated by the introductionof DNA damage if desired. Whether the reduction in RI is mediated bydeletion formation between repeated sequences by an “intra-molecular”mechanism, or mediated by recombination-like events through“inter-molecular” mechanisms is immaterial. The end result is areassortment of the molecules into all possible combinations.

Optionally, the method comprises the additional step of screening thelibrary members of the shuffled pool to identify individual shuffledlibrary members having the ability to bind or otherwise interact, orcatalyze a particular reaction (e.g., such as catalyzing the hydrolysisof a haloalkane).

The polypeptides that are identified from such libraries can be used fortherapeutic, diagnostic, research and related purposes (e.g., catalysts,solutes for increasing osmolarity of an aqueous solution, and the like),and/or can be subjected to one or more additional cycles of shufflingand/or selection.

In another aspect, prior to or during recombination or reassortment,polynucleotides of the invention or polynucleotides generated by themethod described herein can be subjected to agents or processes whichpromote the introduction of mutations into the original polynucleotides.The introduction of such mutations would increase the diversity ofresulting hybrid polynucleotides and polypeptides encoded therefrom. Theagents or processes which promote mutagenesis can include, but are notlimited to: (+)-CC-1065, or a synthetic analog such as(+)-CC-1065-(N-3-Adenine, see Sun and Hurley, 1992); an N-acelylated ordeacetylated 4′-flure-4-aminobiphenyl adduct capable of inhibiting DNAsynthesis (see, for example, van de Poll et al., 1992); or aN-acetylated or deacetylated 4-aminobiphenyl adduct capable ofinhibiting DNA synthesis (see also, van de Poll et al., 1992, pp.751-758); trivalent chromium, a trivalent chromium salt, a polycyclicaromatic hydrocarbon (“PAH”) DNA adduct capable of inhibiting DNAreplication, such as 7-bromomethyl-benz[α]anthracene (“BMA”),tris(2,3-dibromopropyl)phosphate (“Tris-BP”),1,2-dibromo-3-chloropropane (“DBCP”), 2-bromoacrolein (2BA),benzo[a]pyrene-7,8-dihydrodiol-9-10-epoxide (“BPDE”), a platinum(II)halogen salt, N-hydroxy-2-amino-3-methylimidazo[4,5-f]-quinoline(“N-hydroxy-IQ”), andN-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-f]-pyridine(“N-hydroxy-PhIP”). Especially preferred means for slowing or haltingPCR amplification consist of UV light (+)-CC-1065 and(+)-CC-1065-(N-3-Adenine). Particularly encompassed means are DNAadducts or polynucleotides comprising the DNA adducts from thepolynucleotides or polynucleotides pool, which can be released orremoved by a process including heating the solution comprising thepolynucleotides prior to further processing.

In another aspect the invention is directed to a method of producingrecombinant proteins having biological activity by treating a samplecomprising double-stranded template polynucleotides encoding a wild-typeprotein under conditions according to the invention which provide forthe production of hybrid or re-assorted polynucleotides.

The invention also provides for the use of codon primers (containing adegenerate N,N,N sequence) to introduce point mutations into apolynucleotide (e.g., a nucleic acid encoding a phytase of theinvention, or, any phytase), so as to generate a set of progenypolypeptides in which a full range of single amino acid substitutions isrepresented at each amino acid position (Gene Site SaturationMutagenesis™ (GSSM™)). The oligos used are comprised contiguously of afirst homologous sequence, a degenerate N,N,N sequence, and can but notnecessarily a second homologous sequence. The downstream progenytranslational products from the use of such oligos include all possibleamino acid changes at each amino acid site along the polypeptide,because the degeneracy of the N,N,N sequence includes codons for all 20amino acids.

In one aspect, one such degenerate oligo (comprised of one degenerateN,N,G/T cassette) is used for subjecting each original codon in aparental polynucleotide template to a full range of codon substitutions.In another aspect, at least two degenerate N,N,G/T cassettes areused—either in the same oligo or not, for subjecting at least twooriginal codons in a parental polynucleotide template to a full range ofcodon substitutions. Thus, more than one N,N,G/T sequence can becontained in one oligo to introduce amino acid mutations at more thanone site. This plurality of N,N,G/T sequences can be directlycontiguous, or separated by one or more additional nucleotidesequence(s). In another aspect, oligos serviceable for introducingadditions and deletions can be used either alone or in combination withthe codons containing an N,N,G/T sequence, to introduce any combinationor permutation of amino acid additions, deletions, and/or substitutions.

In a particular exemplification, it is possible to simultaneouslymutagenize two or more contiguous amino acid positions using an oligothat contains contiguous N,N,G/T triplets, i.e. a degenerate (N,N,G/T),sequence.

In another aspect, the present invention provides for the use ofdegenerate cassettes having less degeneracy than the N,N,G/T sequence.For example, it may be desirable in some instances to use (e.g. in anoligo) a degenerate triplet sequence comprised of only one N, where saidN can be in the first second or third position of the triplet. Any otherbases including any combinations and permutations thereof can be used inthe remaining two positions of the triplet. Alternatively, it may bedesirable in some instances to use (e.g., in an oligo) a degenerateN,N,N triplet sequence, or an N,N, G/C triplet sequence.

It is appreciated, however, that the use of a degenerate triplet (suchas N,N,G/T or an N,N, G/C triplet sequence) as disclosed in the instantinvention is advantageous for several reasons. In one aspect, thisinvention provides a means to systematically and fairly easily generatethe substitution of the full range of possible amino acids (for a totalof 20 amino acids) into each and every amino acid position in apolypeptide. Thus, for a 100 amino acid polypeptide, the inventionprovides a way to systematically and fairly easily generate 2000distinct species (i.e., 20 possible amino acids per position times 100amino acid positions). It is appreciated that there is provided, throughthe use of an oligo containing a degenerate N,N,G/T or an N,N, G/Ctriplet sequence, 32 individual sequences that code for 20 possibleamino acids. Thus, in a reaction vessel in which a parentalpolynucleotide sequence is subjected to saturation mutagenesis using onesuch oligo, there are generated 32 distinct progeny polynucleotidesencoding 20 distinct polypeptides. In contrast, the use of anon-degenerate oligo in site-directed mutagenesis leads to only oneprogeny polypeptide product per reaction vessel.

This invention also provides for the use of nondegenerate oligos, whichcan optionally be used in combination with degenerate primers disclosed.It is appreciated that in some situations, it is advantageous to usenondegenerate oligos to generate specific point mutations in a workingpolynucleotide. This provides a means to generate specific silent pointmutations, point mutations leading to corresponding amino acid changes,and point mutations that cause the generation of stop codons and thecorresponding expression of polypeptide fragments.

Thus, in one embodiment, each saturation mutagenesis reaction vesselcontains polynucleotides encoding at least 20 progeny polypeptidemolecules such that all 20 amino acids are represented at the onespecific amino acid position corresponding to the codon positionmutagenized in the parental polynucleotide. The 32-fold degenerateprogeny polypeptides generated from each saturation mutagenesis reactionvessel can be subjected to clonal amplification (e.g., cloned into asuitable E. coli host using an expression vector) and subjected toexpression screening. When an individual progeny polypeptide isidentified by screening to display a favorable change in property (whencompared to the parental polypeptide), it can be sequenced to identifythe correspondingly favorable amino acid substitution contained therein.

It is appreciated that upon mutagenizing each and every amino acidposition in a parental polypeptide using saturation mutagenesis asdisclosed herein, favorable amino acid changes may be identified at morethan one amino acid position. One or more new progeny molecules can begenerated that contain a combination of all or part of these favorableamino acid substitutions. For example, if 2 specific favorable aminoacid changes are identified in each of 3 amino acid positions in apolypeptide, the permutations include 3 possibilities at each position(no change from the original amino acid, and each of two favorablechanges) and 3 positions. Thus, there are 3×3×3 or 27 totalpossibilities, including 7 that were previously examined—6 single pointmutations (i.e., 2 at each of three positions) and no change at anyposition.

In yet another aspect, site-saturation mutagenesis can be used togetherwith shuffling, chimerization, recombination and other mutagenizingprocesses, along with screening. This invention provides for the use ofany mutagenizing process(es), including saturation mutagenesis, in aniterative manner. In one exemplification, the iterative use of anymutagenizing process(es) is used in combination with screening.

Thus, in a non-limiting exemplification, polynucleotides andpolypeptides of the invention can be derived by saturation mutagenesisin combination with additional mutagenization processes, such as processwhere two or more related polynucleotides are introduced into a suitablehost cell such that a hybrid polynucleotide is generated byrecombination and reductive reassortment.

In addition to performing mutagenesis along the entire sequence of agene, mutagenesis can be used to replace each of any number of bases ina polynucleotide sequence, wherein the number of bases to be mutagenizedcan be every integer from 15 to 100,000. Thus, instead of mutagenizingevery position along a molecule, one can subject every or a discretenumber of bases (can be a subset totaling from 15 to 100,000) tomutagenesis. In one aspect, a separate nucleotide is used formutagenizing each position or group of positions along a polynucleotidesequence. A group of 3 positions to be mutagenized may be a codon. Themutations can be introduced using a mutagenic primer, containing aheterologous cassette, also referred to as a mutagenic cassette.Preferred cassettes can have from 1 to 500 bases. Each nucleotideposition in such heterologous cassettes be N, A, C, G, T, A/C, A/G, A/T,C/G, C/T, G/T, C/G/T, A/G/T, A/C/T, A/C/G, or E, where E is any basethat is not A, C, G, or T (E can be referred to as a designer oligo).

In a general sense, saturation mutagenesis is comprised of mutagenizinga complete set of mutagenic cassettes (wherein each cassette can beabout 1-500 bases in length) in defined polynucleotide sequence to bemutagenized (wherein the sequence to be mutagenized can be from about 15to 100,000 bases in length). Thus, a group of mutations (ranging from 1to 100 mutations) is introduced into each cassette to be mutagenized. Agrouping of mutations to be introduced into one cassette can bedifferent or the same from a second grouping of mutations to beintroduced into a second cassette during the application of one round ofsaturation mutagenesis. Such groupings are exemplified by deletions,additions, groupings of particular codons, and groupings of particularnucleotide cassettes.

Defined sequences to be mutagenized include a whole gene, pathway, cDNA,an entire open reading frame (ORF), and entire promoter, enhancer,repressor/transactivator, origin of replication, intron, operator, orany polynucleotide functional group. Generally, a “defined sequences”for this purpose may be any polynucleotide that a 15 base-polynucleotidesequence, and polynucleotide sequences of lengths between 15 bases and15,000 bases (this invention specifically names every integer inbetween). Considerations in choosing groupings of codons include typesof amino acids encoded by a degenerate mutagenic cassette.

In one aspect a grouping of mutations can be introduced into a mutageniccassette, this invention specifically provides for degenerate codonsubstitutions (using degenerate oligos) that code for 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 amino acids at eachposition, and a library of polypeptides encoded thereby.

The present invention also includes polynucleotides, wherein the codingsequence for the mature enzyme may be fused in the same reading frame toa polynucleotide sequence which aids in expression and secretion of anenzyme from a host cell, for example, a leader sequence which functionsto control transport of an enzyme from the cell. An enzyme having aleader sequence is an example of a preprotein and may have the leadersequence cleaved by the host cell to form the mature form of the enzyme.The polynucleotides may also encode for a proprotein which isexemplified by a mature protein plus additional 5′ amino acid residues.An otherwise mature protein having a prosequence is exemplified by aproprotein that is an inactive form of the protein. Once the prosequenceis cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention mayencode for a mature enzyme, or for an enzyme having a prosequence or foran enzyme having both a prosequence and a presequence (e.g. leadersequence).

The coding sequences for the phytase enzymes of the present inventionwere identified by preparing E. coli B genomic DNA, for example, andrecovering (via, for example, PCR amplification) from the genomic DNA,DNA encoding phytase activity. Such methods for recovery are well-knownin the art. One means, for example, comprises designing amplificationprimers to recover the coding sequence, amplifying the gene from thegenomic DNA, subcloning the DNA into a vector, transforming theresulting construct into a host strain, and expressing the phytaseenzyme for evaluation. Such procedures are well known in the art andmethods are provided, for example, in Sambrook et al., 1989, which ishereby incorporated by reference in its entirety.

An exemplary enzyme of the present invention was isolated from an E.coli B genomic DNA by the following technique:

-   E. coli B genomic DNA was obtained comercially (Sigma: Catalog #    D-2001, St. Louis, N.J.). The following primers were used to amplify    the gene directly from the genomic DNA:-   5′ primer gtttctgaattcaaggaggaatttaaATGAAAGCGATCTTAATCCCATT (SEQ ID    NO:3); and-   3′ primer gtttctggatccTTACAAACTGCACGCCGGTAT (SEQ ID NO:4)-   Pfu polymerase was used according to manufacturers protocol    (Stratagene Cloning Systems, Inc., La Jolla, Calif.).

PCR product and pQE60 vector (Qiagen) were both digested with EcoRI andBglII restriction endonucleases (New England Biolabs) according tomanufacturers protocols. Ligation and transformation into, andexpression in M15 pREP4 host cells (Qiagen) yields c-term 6×-His taggedprotein.

The isolated nucleic acid sequences and other enzymes may then bemeasured for retention of biological activity characteristic to theenzyme of the present invention, for example, in an assay for detectingenzymatic phytase activity (Food Chemicals Codex, 4^(th) Ed.). Suchenzymes include truncated forms of phytase, and variants such asdeletion and insertion variants.

An in vitro example of such an assay is the following assay for thedetection of phytase activity: Phytase activity can be measured byincubating 150 μl of the enzyme preparation with 600 μl of 2 mM sodiumphytate in 100 mM Tris HCl buffer pH 7.5, supplemented with 1 mM CaCl₂for 30 minutes at 37° C. After incubation the reaction is stopped byadding 750 μl of 5% trichloroacetic acid. Phosphate released wasmeasured against phosphate standard spectrophotometrically at 700 nmafter adding 1500 μl of the color reagent (4 volumes of 1.5% ammoniummolybdate in 5.5% sulfuric acid and 1 volume of 2.7% ferrous sulfate;Shimizu, 1992). One unit of enzyme activity is defined as the amount ofenzyme required to liberate one μmol Pi per min under assay conditions.Specific activity can be expressed in units of enzyme activity per mg ofprotein.

The enzyme of the present invention has enzymatic activity with respectto the hydrolysis of phytate to inositol and free phosphate.

The enzymes and polynucleotides of the present invention can be providedin an isolated form, and can be purified to homogeneity. The phytasepolypeptide of the invention can be obtained using any of severalstandard methods. For example, phytase polypeptides can be produced in astandard recombinant expression system (see below), chemicallysynthesized (this approach may be limited to small phytase peptidefragments), or purified from organisms in which they are naturallyexpressed. Serviceable recombinant expression methods include the use ofmammalian hosts, microbial hosts, and plant hosts.

The recombinant expression of the instant phytase molecules may beachieved in combination with one or more additional molecules such as,for example, other enzymes. This approach is serviceable for producingcombination products, such as a plant or plant part that contains theinstant phytase molecules as well as one or more additionalmolecules—preferably said phytase molecules and said additionalmolecules are serviceable in a combination treatment. The resultingrecombinantly expresssed molecules may be used in homogenized and/orpurified form or alternatively in relatively unpurified form (e.g. asconsumable plant parts that are serviceable when admixed with otherfoodstuffs for catalyzing the degredation of phytate).

In sum, in a non-limiting embodiment, the present invention provides arecombinant enzyme expressed in a host. In another non-limitingembodiment, the present invention provides a substantially pure phytaseenzyme. Thus, an enzyme of the present invention may be a recombinantenzyme, a natural enzyme, or a synthetic enzyme, preferably arecombinant enzyme.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention, and the productionof enzymes of the invention by recombinant techniques.

Host cells are genetically engineered (e.g. transduced or transformed ortransfected) with the vectors containing the polynucleotides of thisinvention. Such vectors may be, for example, a cloning vector or anexpression vector. The vector may be, for example, in the form of aplasmid, a viral particle, a phage, a prion, etc. The engineered hostcells can be cultured in conventional nutrient media modified asappropriate for activating promoters, &/or selecting transformants oramplifying the genes of the present invention. The culture conditions,such as temperature, pH and the like, are those previously used with thehost cell selected for expression, and will be apparent to theordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing enzymes by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing an enzyme. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Inclusive in this meaning is the use of blunt-ended molecules whichcould be generated by the use of restriction digestion as well asrestriction digestion-independent means. Alternatively, the insert maybe incorporated into a vector by so called “ligase-independent” means.In a particular aspect, a “ligase-independent” means is exemplified bythe use of topoisomerase-mediated ligation at room temperature, forexample according to the commercially available kit termed TOPO-TACloning® (Invitrogen Corporation, Carlsbad, Calif.). Alteranativeenzymes, including isomers of topoisomerase as well as more distantlyrelated recombination enzymes (e.g. recombinases), may also beserviceable for mediating this type of “ligase-independent”incorporation. In another particular aspect, a “ligase-independent”means is exemplified by the use host repair mechanisms. Such proceduresand others are deemed to be within the scope of those skilled in theart.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: an LTR or SV40 promoter, an E. coli. lac or trp, a phagelambda PL promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

Exemplary organisms for expressing polypeptides of the invention can beS. pombe, S. cerevisiae, Pichia sp., e.g., P. pastoris, E. coli,Streptomyces sp., Bacillus sp. and Lactobacillus sp. Exemplary hosts forexpressing polypeptides and nucleic acids of the invention, and topractice the methods of the invention, include bacterial cells, such asE. coli, Streptomyces, Bacillus ceres, Bacillus subtilis; fungal cells,such as yeast; insect cells such as Drosophila 52 and Spodoptera Sf9;animal cells such as CHO, COS or Bowes melanoma; adenoviruses; plantcells, etc. The selection of an appropriate host is deemed to be withinthe scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence. Oneor more additional inserts may also be incorporated that lead toexpression of one or more aditional molecules, such as another phytaseor a protease enzyme, preferably said one or more additional moleculesare serviceable in combination with the instant phytase in a combinationtreatment.

Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. “Plasmids” aredesignated by a lower case p preceded and/or followed by capital lettersand/or numbers. The starting plasmids herein are either commerciallyavailable, publicly available on an unrestricted basis, or can beconstructed from available plasmids in accord with published procedures.In addition, equivalent plasmids to those described are known in the artand will be apparent to the ordinarily skilled artisan.

The following vectors are provided by way of example; Bacterial: pQE70,pQE60, pQE-9 (Qiagen), pBluescript II® (Stratagene); pTRC99a, pKK223-3,pDR540, pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5 (Stratagene) pSVK3,pBPV, pMSG, pSVLSV40 (Pharmacia). However, any other plasmids or othervectors may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, 1986).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the enzymes of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described (e.g. Sambrook et al., 1989, thedisclosure of which is hereby incorporated by reference).

Transcription of the DNA encoding the enzymes of the present inventionby higher eukaryotes is increased by inserting an enhancer sequence intothe vector. Enhancers are cis-acting elements of DNA, usually about from10 to 300 bp that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin bp 100 to 270, a cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP 1 gene, and a promoter derived from a highly-expressed gene todirect transcription of a downstream structural sequence. Such promoterscan be derived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), Å-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated enzyme. Optionally, the heterologoussequence can encode a fusion enzyme including an N-terminalidentification peptide imparting desired characteristics, e.g.,stabilization or simplified purification of expressed recombinantproduct.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Bacillus cereus,Salmonella typhimurium and various species within the generaStreptomyces, Bacillus and Staphylococcus, although others may also beemployed as a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell known to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, as described (Gluzman,1981), and other cell lines capable of expressing a compatible vector,for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalianexpression vectors will comprise an origin of replication, a suitablepromoter and enhancer, and also any necessary ribosome binding sites,polyadenylation site, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking nontranscribed sequences. DNAsequences derived from the SV40 splice, and polyadenylation sites may beused to provide the required nontranscribed genetic elements.

The enzyme can be recovered and purified from recombinant cell culturesby methods including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The enzymes of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the enzymes of the present invention may beglycosylated or may be non-glycosylated. Enzymes of the invention may ormay not also include an initial methionine amino acid residue.

In a preferred embodiment, the enzyme of the present invention is aphytase enzyme which is stable to heat and is heat resistant andcatalyzes the enzymatic hydrolysis of phytate, i.e., the enzyme is ableto renature and regain activity after a brief (i.e., 5 to 30 seconds),or longer period, for example, minutes or hours, exposure totemperatures of up to about 50° C. or slightly above 50° C.

The present invention is further described with reference to theexamples contained herein; however, it is to be understood that thepresent invention is not limited to such examples. All parts or amounts,unless otherwise specified, are by weight.

The present invention also provides an isolated variant phytasepolynucleotide, or an oligonucleotide portion thereof comprising amutation as disclosed herein. As used herein, the term “isolated” or“purified,” when used in reference to a polynucleotide, oligonucleotide,or polypeptide, means that the material is in a form other than that inwhich it normally is found in nature. Thus, where a polynucleotide orpolypeptide occurs in a cell in nature, an isolated polynucleotide orpurified polypeptide can be one that separated, at least in part, fromthe materials with which it is normally associated with. In general, anisolated polynucleotide or a purified polypeptide is present in a formin which it constitutes at least about 5 to 10% of a composition,usually 20% to 50% of a composition, particularly about 50% to 75% of acomposition, and preferably about 90% to 95% or more of a composition.Methods for isolating a polynucleotide or polypeptide are well known androutine in the art.

As part of or following isolation, a polynucleotide can be joined toother polynucleotides, such as DNA molecules, for example, formutagenesis studies, to form fusion proteins, or for propagation orexpression of the polynucleotide in a host. The isolatedpolynucleotides, alone or joined to other polynucleotides, such asvectors, can be introduced into host cells, in culture or in wholeorganisms. Such polynucleotides, when introduced into host cells inculture or in whole organisms, nevertheless are considered “isolated”because they are not in a form in which they exist in nature. Similarly,the polynucleotides, oligonucleotides, and polypeptides can be presentin a composition such as a media formulation (solutions for introductionof polynucleotides, oligonucleotides, or polypeptides, for example, intocells or compositions or solutions for chemical or enzymatic reactionswhich are not naturally occurring compositions) and, therein remainisolated polynucleotides, oligonucleotides, or polypeptides within themeaning of that term as it is employed herein. An isolatedpolynucleotide can be a polynucleotide that is not immediatelycontiguous with nucleotide sequences with which it is immediatelycontiguous in a genome or other naturally occurring cellular DNAmolecule in nature. Thus, a recombinant polynucleotide, which cancomprise a polynucleotide incorporated into a vector, an autonomouslyreplicating plasmid, or a virus; or into the genomic DNA of a prokaryoteor eukaryote, which does not normally express a particular polypeptide.

As used herein, the term “polynucleotide” or “oligonucleotide” or“nucleotide sequence” or the like refers to a polymer of two or morenucleotides or nucleotide analogs. The polynucleotide can be aribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecule, and canbe single stranded or double stranded DNA or RNA, or a double strandedDNA:RNA hybrid. A polynucleotide or oligonucleotide can contain one ormore modified bases, for example, inosine or a tritylated base. Thebonds linking the nucleotides in a polymer generally are phosphodiesterbonds, but can be other bonds routinely used to link nucleotidesincluding, for example, phosphorothioate bonds, thioester bonds, and thelike. A polynucleotide also can be a chemically, enzymatically ormetabolically modified form.

As used herein, the term “mutant or variant polynucleotide” means anucleotide sequence that has one or a few nucleotide changes as comparedto the nucleotide sequence set forth as SEQ ID NO:1, 7 or 9, forexample. The nucleotide change can be a deletion, insertion orsubstitution, and can be silent such that there is no change in thereading frame of a polypeptide encoded by the wild-type polynucleotide,or can be a change that results in an amino acid change or in theintroduction of a STOP codon into the polynucleotide, or a change in anucleotide sequence involved in transcription or translation of thepolynucleotide, for example, a change that results in altered splicingof a gene transcript into an mRNA.

For convenience of discussion and for use as a frame of reference, thephytase nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:7 isreferred to as a “wild type” polynucleotide or “wild type” genesequence, and, similarly, the polypeptide set forth as SEQ ID NO:2 orSEQ ID NO:8 (E. coli appA wild type phytase) is referred to as a wildtype phytase polypeptide.

Examples of a variant phytase polynucleotide sequence include sequencessubstantially as set forth in SEQ ID NO:7, wherein the polynucleotidehas a nucleotide sequence as set forth in a) SEQ ID NO:9; b) SEQ ID NO:9wherein all Ts are Us (RNA); wherein the expression of thephytase-encoding nucleic acid leads to the production of saidsubstantially pure phytase enzyme; and c) SEQ ID NO:7, wherein 389 is G;390 is A; nucleotide 437 is T; 438 is G; 439 is G; 470 is C; 72 is T;476 is T; 477 is G; 478 is T; 689 is G; 690 is A; 691 is G; 728 is T;729 is A; 730 is T; 863 is T; 864 is G; 1016 is G, or any combinationthereof. More specifically, with respect to part c), the inventionprovides a nucleotide sequence substantially identical to SEQ ID NO:7,and having a modified nucleotide sequence selected from nucleotide 389is G and 390 is A; nucleotide 437 is T, 438 is G and 439 is G; 470 is Cand 472 is T; 476 is T, 477 is G, and 478 is T; 689 is G, 690 is A and691 is G; 728 is T, 729 is A, and 730 is T; 863 is T and 864 is G; 1016is G, or any combination thereof.

Examples of a variant phytase polynucleotide of the invention alsoinclude a polynucleotide that encodes a polypeptide having substantiallyas set forth in SEQ ID NO:8, but having an W68E, Q84W, A95P, K97C,S168E, R181Y, N226C, Y277D or any combination thereof and retain phytaseactivity.

Additional examples of mutant polynucleotides of the invention includepolynucleotide sequences that selectively hybridize to the complementsof the polynucleotide sequences, or oligonucleotide portions thereof, asdisclosed herein, under highly stringent hybridization conditions, e.g.,hybridization to filter-bound DNA in 0.5M NaHPO₄, 7% sodium dodecylsulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at68° C. (Ausubel et al., Current Protocols in Molecular Biology, (GreenPublishing Associates, Inc., and John Wiley & Sons, Inc., New York1989), and supplements; see p. 2.10.3; Sambrook et al., MolecularCloning: A laboratory manual (Cold Spring Harbor Laboratory Press,1989), which are incorporated herein by reference), as well aspolynucleotides that encode a phytase polypeptide substantially as setforth in SEQ ID NO:8, but having one or more mutations; or an RNAcorresponding to such a polynucleotide (e.g., SEQ ID NO:9).

In alternative aspects, the invention provides polynucleotide orpolypeptide sequences having 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%,98.5%, 99%, 99.5%, or more, or complete (100%) sequence identity (i.e.,homology) (this encompassing the term “substantially identical”) to aphytase-encoding polynucleotide or phytase polypeptide of the invention,including: SEQ ID NO:1 (a phytase-encoding polynucleotide); SEQ ID NO:2(a phytase polypeptide); SEQ ID NO:9 (a phytase-encodingpolynucleotide); SEQ ID NO:10 (a phytase polypeptide); a nucleic acidhaving sequence as set forth in SEQ ID NO:7 and wherein nucleotide 389is G; 390 is A; 437 is T; 438 is G; 439 is G; 470 is C; 472 is T; 476 isT; 477 is G; 478 is T; 689 is G; 690 is A; 691 is G; 728 is T; 729 is A;730 is T; 863 is T; 864 is G; or, 1016 is G, or, any combinationthereof, wherein the polynucleotide encodes a phytase; a nucleic acidhaving sequence as set forth in SEQ ID NO:7 and wherein nucleotide 389is G; 390 is A; 437 is T; 438 is G; 439 is G; 470 is C; 472 is T; 476 isT; 477 is G; 478 is T; 689 is G; 690 is A; 691 is G; 728 is T; 729 is A;730 is T; 863 is T; 864 is G; and 1016 is G; a nucleic acid encoding apolypeptide having sequence as set forth in SEQ ID NO:8 and having oneor more amino acid modifications selected from W68E, Q84W, A95P, K97C,S168E, R181 Y, N226C, Y277D, or any combination thereof, wherein thepolypeptide has phytase activity; a nucleic acid encoding a polypeptidehaving sequence as set forth in SEQ ID NO:8 and having the amino acidmodifications W68E, Q84W, A95P, K97C, S168E, R181Y, N226C, Y277D,wherein the polypeptide has phytase activity; or other phytases, forexample, the E. coli appA “wild type” phytase-encoding SEQ ID NO:7, or,a polypeptide sequence of SEQ ID NO:2 or the E. coli appA “wild type”phytase SEQ ID NO:8.

Sequence identity can be measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison Wis. 53705).

A polynucleotide or oligonucleotide portion thereof of the invention canbe useful, for example, as a probe or as a primer for an amplificationreaction. Reference to an “oligonucleotide portion” of a polynucleotidemeans a nucleotide sequence of the variant or mutant polynucleotide thatis less than the full length polynucleotide. Generally, anoligonucleotide useful as a probe or a primer contains at least about 10nucleotides, and usually contains about 15 to 30 nucleotides or more(see, for example, Tables 1 and 2). Polynucleotides and oligonucleotidescan be prepared by any suitable method, including, for example, byrestriction enzyme digestion of an appropriate polynucleotide, by directchemical synthesis using a method such as the phosphotriester method(Narang et al., 1979, Meth. Enzymol., 68:90-99); the phosphodiestermethod (Brown et al., 1979, Meth. Enzymol., 68:109-151); thediethylphosphoramidite method (Beaucage et al., 1981, Tetrahedron Lett.,22:1859-1862); the triester method (Matteucci et al., 1981, J. Am. Chem.Soc., 103:3185-3191), including by automated synthesis methods; or by asolid support method (see, for example, U.S. Pat. No. 4,458,066). Inaddition, a polynucleotide or oligonucleotide can be prepared usingrecombinant DNA methods as disclosed herein or otherwise known in theart.

An oligonucleotide of the invention can include a portion of a phytasepolynucleotide, including, for example, a sequence substantiallyidentical to that of SEQ ID NO:7, except wherein nucleotide wherein 389is G; 390 is A; 437 is T; 438 is G; 439 is G; 470 is C; 472 is T; 476 isT; 477 is G; 478 is T; 689 is G; 690 is A; 691 is G; 728 is T; 729 is A;730 is T; 863 is T; 864 is G; or, 1016 is G, or wherein theoligonucleotide contains a combination of such substitutions withrespect to SEQ ID NO:7. Thus, as disclosed herein, the oligonucleotidecan be any length and can encompass one or more of the above mutations.

An oligonucleotide of the invention can selectively hybridize to amutant phytase polynucleotide sequence as disclosed herein. As usedherein, “selectively hybridize” refers to the ability of anoligonucleotide (or polynucleotide) probe to hybridize to a mutantpolynucleotide, but not substantially to a wild-type sequence.Hybridization conditions that allow for selective hybridization can beobtained by varying the stringency of the hybridization conditions, asdescribed above, and will depend, in part, on the length of the probe,the relative G:C content, the salt concentration, and the like (seeSambrook et al., supra, 1989). Hybridization conditions that are highlystringent conditions include, for example, washing in 6×SSC/0.05% sodiumpyrophosphate at about 37° C. (for 14 nucleotide DNA probe), about 48°C. (for 17 nucleotide probe), about 55° C. (for a 20 nucleotide probe),and about 60° C. (for a 23 nucleotide probe).

An oligonucleotide of the invention can be used as a probe to screen fora particular variant or mutant of interest. In addition, theoligonucleotides of the invention include an antisense molecule, whichcan be useful, for example, in polynucleotide regulation andamplification reactions of polynucleotide sequences, including mutantphytase polynucleotide sequences. Further, such oligonucleotides can beused as part of ribozyme or triple helix sequence for phytase generegulation. Still further, such oligonucleotides can be used as acomponent of diagnostic method, whereby the level of phytase transcriptcan be determined. Further, such oligonucleotides can be used, forexample, to screen for and identify phytase homologs from other species.

The term “primer” or “PCR primer” refers to an isolated natural orsynthetic oligonucleotide that can act as a point of initiation of DNAsynthesis when placed under conditions suitable for primer extension.Synthesis of a primer extension product is initiated in the presence ofnucleoside triphosphates and a polymerase in an appropriate buffer at asuitable temperature. A primer can comprise a plurality of primers, forexample, where there is some ambiguity in the information regarding oneor both ends of the target region to be synthesized. For instance, if anucleic acid sequence is determined from a protein sequence, a primergenerated to synthesize nucleic acid sequence encoding the proteinsequence can comprise a collection of primers that contains sequencesrepresenting all possible codon variations based on the degeneracy ofthe genetic code. One or more of the primers in this collection will behomologous with the end of the target sequence or a sequence flanking atarget sequence. Likewise, if a conserved region shows significantlevels of polymorphism in a population, mixtures of primers can beprepared that will amplify adjacent sequences.

During PCR amplification, primer pairs flanking a target sequence ofinterest are used to amplify the target sequence. A primer pairtypically comprises a forward primer, which hybridizes to the 5′ end ofthe target sequence, and a reverse primer, which hybridizes to the 3′end of the target sequence. A primer pair of the invention includes atleast one forward primer and at least one reverse primer that allows forgeneration of an amplification product, which can be a long rangephytase-specific amplification product or a nested amplification productof such an amplification product, including a forward and reverse primerprovided that the forward primer is 5′ (or upstream) of the reverseprimer with reference to a target polynucleotide sequence, and that theprimers are in sufficient proximity such that an amplification productcan be generated.

Nucleic acid sequences that encode a fusion protein can be produced andcan be operatively linked to expression control sequences. Such fusionproteins and compositions are useful in the development of antibodies orto generate and purify peptides and polypeptides of interest. As usedherein, the term “operatively linked” refers to a juxtaposition, whereinthe components so described are in a relationship permitting them tofunction in their intended manner. For example, an expression controlsequence operatively linked to a coding sequence is ligated such thatexpression of the coding sequence is achieved under conditionscompatible with the expression control sequences, whereas twooperatively linked coding sequences can be ligated such that they are inthe same reading frame and, therefore, encode a fusion protein.

As used herein, the term “expression control sequences” refers tonucleic acid sequences that regulate the expression of a nucleic acidsequence to which it is operatively linked. Expression control sequencesare operatively linked to a nucleic acid sequence when the expressioncontrol sequences control and regulate the transcription and, asappropriate, translation of the nucleic acid sequence. Thus, expressioncontrol sequences can include appropriate promoters, enhancers,transcription terminators, a start codon (i.e., ATG) in front of aprotein-encoding gene, splicing signals for introns, maintenance of thecorrect reading frame of that gene to permit proper translation of themRNA, and STOP codons. Control sequences include, at a minimum,components whose presence can influence expression, and can also includeadditional components whose presence is advantageous, for example,leader sequences and fusion partner sequences. Expression controlsequences can include a promoter.

A polynucleotide of the invention can comprise a portion of arecombinant nucleic acid molecule, which, for example, can encode afusion protein. The polynucleotide, or recombinant nucleic acidmolecule, can be inserted into a vector, which can be an expressionvector, and can be derived from a plasmid, a virus or the like. Theexpression vector generally contains an origin of replication, apromoter, and one or more genes that allow phenotypic selection oftransformed cells containing the vector. Vectors suitable for use in thepresent invention include, but are not limited to the T7-basedexpression vector for expression in bacteria (Rosenberg, et al., Gene56:125, 1987), the pMSXND expression vector for expression in mammaliancells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988);baculovirus-derived vectors for expression in insect cells; and thelike.

The choice of a vector will also depend on the size of thepolynucleotide sequence and the host cell to be employed in the methodsof the invention. Thus, the vector used in the invention can beplasmids, phages, cosmids, phagemids, viruses (e.g., retroviruses,parainfluenzavirus, herpesviruses, reoviruses, paramyxoviruses, and thelike), or selected portions thereof (e.g., coat protein, spikeglycoprotein, capsid protein). For example, cosmids and phagemids aretypically used where the specific nucleic acid sequences to be analyzedor modified is large because these vectors are able to stably propagatelarge polynucleotides. Cosmids and phagemids are particularly suited forthe expression or manipulation of the phytase polynucleotide of SEQ IDNO:1 or 7 or a mutant phytase polynucleotide as in SEQ ID NO:9.

In yeast, a number of vectors containing constitutive or induciblepromoters can be used (see Ausubel et al., supra, 1989; Grant et al.,Meth. Enzymol. 153:516-544, 1987; Glover, DNA Cloning, Vol. II, IRLPress, Washington D.C., Ch. 3, 1986; and Bitter, Meth. Enzymol.152:673-684, 1987; and The Molecular Biology of the Yeast Saccharomyces,Eds. Strathern et al., Cold Spring Harbor Press, Vols. I and II, 1982).A constitutive yeast promoter such as ADH or LEU2 or an induciblepromoter such as GAL can be used (“Cloning in Yeast,” Ch. 3, Rothstein,In “DNA Cloning” Vol. 11, A Practical Approach, ed. Glover, IRL Press,1986). Alternatively, vectors can be used which promote integration offoreign DNA sequences into the yeast chromosome. The construction ofexpression vectors and the expression of genes in transfected cellsinvolves the use of molecular cloning techniques also well known in theart (see Sambrook et al., supra, 1989; Ausubel et al., supra, 1989).These methods include in vitro recombinant DNA techniques, synthetictechniques and in vivo recombination/genetic recombination.

A polynucleotide or oligonucleotide can be contained in a vector and canbe introduced into a cell by transformation or transfection of the cell.By “transformation” or “transfection” is meant a permanent (stable) ortransient genetic change induced in a cell following incorporation ofnew DNA (i.e., DNA exogenous to the cell). Where the cell is a mammaliancell, a permanent genetic change is generally achieved by introductionof the DNA into the genome of the cell.

A transformed cell or host cell can be any prokaryotic or eukaryoticcell into which (or into an ancestor of which) has been introduced, bymeans of recombinant DNA techniques, a polynucleotide sequence of theinvention or fragment thereof. Transformation of a host cell can becarried out by conventional techniques as are well known to thoseskilled in the art. Where the host is prokaryotic, such as E. coli,competent cells which are capable of DNA uptake can be prepared fromcells harvested after exponential growth phase and subsequently treatedby the CaCl₂ method by procedures well known in the art, or using MgCl₂or RbCl. Transformation can also be performed after forming a protoplastof the host cell or by electroporation.

When the host is a eukaryote, such methods of transfection include theuse of calcium phosphate co-precipitates, conventional mechanicalprocedures such as microinjection, electroporation, insertion of aplasmid encased in liposomes, or the use of virus vectors, or othermethods known in the art. One method uses a eukaryotic viral vector,such as simian virus 40 (SV40) or bovine papillomavirus, to transientlyinfect or transform eukaryotic cells and express the protein.(Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed.,1982). Preferably, a eukaryotic host is utilized as the host cell asdescribed herein. The eukaryotic cell can be a yeast cell (e.g.,Saccharomyces cerevisiae), or can be a mammalian cell, including a humancell.

A variety of host-expression vector systems can be utilized to express aphytase polynucleotide sequence such as SEQ ID NO:1 or SEQ ID NO:7, acoding sequence of SEQ ID NO:1 or a mutant phytase polynucleotide suchas SEQ ID NO:9. Such host-expression systems represent vehicles by whichthe nucleotide sequences of interest can be produced and subsequentlypurified, and also represent cells that, when transformed or transfectedwith the appropriate nucleotide coding sequences, can express a protein,including a variant or mutant polypeptide or peptide portion thereof insitu. Such cells include, but are not limited to, microorganisms such asbacteria (e.g., E. coli, B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining a polynucleotide, or oligonucleotide portion thereof (wildtype, variant or other mutant); yeast (e.g., Saccharomyces, Pichia)transformed with recombinant yeast expression vectors containing apolynucleotide, or oligonucleotide portions thereof (wild type, variantor other mutant); insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing a polynucleotide, oroligonucleotide portion thereof (wild type, variant or other mutant);plant cell systems infected with recombinant virus expression vectors(e.g., cauliflower mosaic virus or tobacco mosaic virus) or transformedwith recombinant plasmid expression vectors (e.g., Ti plasmid)containing a mutant polynucleotide, or oligonucleotide portion thereof;or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the phytaseprotein (wild type, variant or other phytase mutant) being expressed.For example, when a large quantity of such a protein is to be produced,for the generation of antibodies, or to screen peptide libraries,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified can be desirable. Such vectorsinclude, but are not limited to, the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which a phytasepolynucleotide, or oligonucleotide portion thereof (wild type, variantor other mutant) can be ligated individually into the vector in framewith the lac Z coding region so that a fusion protein is produced; pINvectors (Inouye and Inouye, Nucl. Acids Res. 13:3101-3109, 1985; VanHeeke and Schuster, J. Biol. Chem. 264:5503-5509, 1989); and the like.pGEX vectors can also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned phytaseprotein, variant or mutant can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. A phytase polynucleotide, oroligonucleotide portion thereof can be cloned individually intonon-essential regions (for example the polyhedrin gene) of the virus andplaced under control of an AcNPV promoter (for example the polyhedrinpromoter). Successful insertion of a phytase polynucleotide, oroligonucleotide portion thereof will result in inactivation of thepolyhedrin gene and production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat coded for by the polyhedrin gene).These recombinant viruses are then used to infect Spodoptera frugiperdacells in which the inserted gene is expressed (see Smith et al., 1983,J. Virol. 46:584; U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, a phytase polynucleotide, or oligonucleotide portion thereof,can be ligated to an adenovirus transcription/translation controlcomplex, e.g., the late promoter and tripartite leader sequence. Thischimeric gene can then be inserted in the adenovirus genome by in vitroor in vivo recombination. Insertion in a non-essential region of theviral genome such as the E1 or E3 region results in a recombinant virusthat is viable and capable of expressing a phytase protein (e.g.,wild-type, variants or mutants thereof) in infected hosts (Logan andShenk, Proc. Natl. Acad. Sci., USA 81:3655-3659, 1984). Specificinitiation signals can also be required for efficient translation of aninserted phytase sequence. These signals include the ATG initiationcodon and adjacent sequences. Where an entire polynucleotide, includingits own initiation codon and adjacent sequences, is inserted into theappropriate expression vector, no additional translational controlsignals can be needed. However, where only a portion of a sequence isinserted, exogenous translational control signals, including, forexample, an ATG initiation codon, must be provided. Furthermore, theinitiation codon must be in phase with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression can be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, and the like (see Bittneret al., Meth. Enzymol. 153:516-544, 1987).

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes theexpressed polypeptide in a specific fashion. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cell lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein being expressed. To this end, eukaryotic hostcells which possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of thepolypeptide can be used. Such mammalian host cells include, but are notlimited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, W138, and thelike.

For long term, high yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express aprotein, including wild-type, variants or mutants of phytase, can beengineered. Rather than using expression vectors which contain viralorigins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoterand/or enhancer sequences, transcription terminators, polyadenylationsites, and the like), and a selectable marker. Following theintroduction of the foreign DNA, engineered cells can be grown for 1-2days in an enriched media, then switched to selective media. Theselectable marker in the recombinant plasmid confers resistance to theselection and allows cells to stably integrate the plasmid into theirchromosomes and grow to form foci, which can be cloned and expanded intocell lines. This method can advantageously be used to engineer celllines that express a phytase variant or mutant polypeptide. Suchengineered cell lines can be particularly useful in screening andevaluation of compounds that affect the endogenous activity of a variantor mutant phytase polypeptide. Such engineered cell lines also can beuseful to discriminate between factors that have specific vs.non-specific effects. In particular, mutant cell lines should lack keyfunctions, and various mutations can be used to identify key functionaldomains using in vivo assays.

A number of selection systems can be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223,1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska andSzybalski, Proc. Natl. Acad. Sci. USA 48:2026, 1962), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817, 1980) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al., Proc.Natl. Acad. Sci. USA 77:3567, 1980; O'Hare et al., Proc. Natl. Acad.Sci. USA 78:1527, 1981); gpt, which confers resistance to mycophenolicacid Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072, 1981); neo,which confers resistance to the aminoglycoside G-418 (Colberre-Garapinet al., J. Mol. Biol. 150:1, 1981); and hygro, which confers resistanceto hygromycin (Santerre et al., Gene 30:147, 1984) genes. Accordingly,the invention provides a vector that contains a mutant phytasepolynucleotide, or oligonucleotide portion thereof, or one or moreprimers or their complements, including an expression vector thatcontains any of the foregoing sequences operatively associated with aregulatory element that directs the expression of a coding sequence orprimer; and also provides a host cell that contains any of the foregoingsequences, alone or operatively associated with a regulatory element,which can directs expression of a polypeptide encoded thepolynucleotide, as appropriate.

A homolog of a mutant phytase polynucleotide sequence can be isolated byperforming a polymerase chain reaction (PCR; see U.S. Pat. No.4,683,202, which is incorporated herein by reference) using twooligonucleotide primers, including degenerate primer pools designed onthe basis of the amino acid sequences of a phytase polypeptide such asthat set forth in SEQ ID NO:8 (E. coli appA “wild type” phytase) or anexemplary phytase of the invention, as disclosed herein. The templatefor the reaction can be cDNA obtained by reverse transcription of mRNAprepared from organisms known to express a phytase enzyme or homologue.The PCR product can be subcloned and sequenced or manipulated in anynumber of ways (e.g., further manipulated by nested PCR) to insure thatthe amplified sequences represent the sequences of a phytase or mutantpolynucleotide sequence. The PCR fragment can then be used to isolate afull length cDNA clone (including clones containing a mutantpolynucleotide sequence) by labeling the amplified fragment andscreening a nucleic acid library (e.g., a bacteriophage cDNA library).Alternatively, the labeled fragment can be used to screen a genomiclibrary (for review of cloning strategies, see, for example, Sambrook etal., supra, 1989; Ausubel et al., supra, 1989).

Phytase polypeptides that have been modified from the wild-type aminoacid sequence, include substitutions of amino acid residues, forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; amino acids with uncharged polar head groups having similarhydrophilicity values include the following: leucine, isoleucine,valine, glycine, alanine, asparagine, glutamine, serine, threonine,phenylalanine and tyrosine. In many cases, however, a nucleotidesubstitution can be silent, resulting in no change in the encodedpolypeptide.

Mutant phytase polypeptides and peptide portions thereof that aresubstantially identical to the phytase polypeptide SEQ ID NO:2 or SEQ IDNO:8 (E. coli appA “wild type” phytase) or peptide portions thereof areencompassed within the scope of the invention.

Synthetic polypeptides or peptides can be prepared by chemicalsynthesis, for example, solid-phase chemical peptide synthesis methods,which are well known (see, for example, Merrifield, J. Am. Chem. Soc.,85:2149-2154, 1963; Stewart and Young, Solid Phase Peptide Synthesis,Second ed., Pierce Chemical Co., Rockford, Ill., pp. 11-12), and havebeen employed in commercially available laboratory peptide design andsynthesis kits (Cambridge Research Biochemicals). Such commerciallyavailable laboratory kits have generally utilized the teachings ofGeysen et al., Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and providefor synthesizing peptides upon the tips of a multitude of rods or pins,each of which is connected to a single plate. When such a system isutilized, a plate of rods or pins is inverted and inserted into a secondplate of corresponding wells or reservoirs, which contain solutions forattaching or anchoring an appropriate amino acid to the tips of the pinsor rods. By repeating such a process step, i.e., inverting and insertingthe tips of the rods or pins into appropriate solutions, amino acids arebuilt into desired peptides.

A number of available FMOC peptide synthesis systems are available. Forexample, assembly of a polypeptide or fragment can be carried out on asolid support using an Applied Biosystems, Inc., Model 431A automatedpeptide synthesizer. Such equipment provides ready access to thepeptides of the invention, either by direct synthesis or by synthesis ofa series of fragments that can be coupled using other known techniques.Accordingly, methods for the chemical synthesis of polypeptides andpeptides are well-known to those of ordinary skill in the art, e.g.,peptides can be synthesized by solid phase techniques, cleaved from theresin and purified by preparative high performance liquid chromatography(see, e.g., Creighton, 1983, Proteins: Structures and MolecularPrinciples, W.H. Freeman & Co., N.Y., pp. 50-60). The composition of thesynthetic peptides can be confirmed by amino acid analysis orsequencing; e.g., using the Edman degradation procedure (see e.g.,Creighton, 1983, supra at pp. 34-49). Thus, fragments of the phytasepolypeptide, variant, or mutant can be chemically synthesized.

In one aspect of the invention, a method for producing an phytaseenzyme, such as those shown in FIG. 1, is provided. The method includesgrowing a host cell which contains a polynucleotide encoding the enzyme(e.g., SEQ ID NO:1, 7 or 9), under conditions which allow the expressionof the nucleic acid, and optionally isolating the enzyme encoded by thenucleic acid. Methods of culturing the host cell are described in theExamples and are known by those of skill in the art.

In a particular embodiment, the present invention provides for theexpression of phytase in transgenic plants or plant organs and methodsfor the production thereof. DNA expression constructs are provided forthe transformation of plants with a gene encoding phytase under thecontrol of regulatory sequences which are capable of directing theexpression of phytase. These regulatory sequences include sequencescapable of directing transcription in plants, either constitutively, orin stage and/or tissue specific manners.

The manner of expression depends, in part, on the use of the plant orparts thereof. The transgenic plants and plant organs provided by thepresent invention may be applied to a variety of industrial processeseither directly, e.g. in animal feeds or alternatively, the expressedphytase may be extracted and if desired, purified before application.Alternatively, the recombinant host plant or plant part may be useddirectly. In a particular aspect, the present invention provides methodsof catalyzing phytate-hydrolyzing reactions using seeds containingenhanced amounts of phytase. The method involves contacting transgenic,non-wild type seeds, preferably in a ground or chewed form, withphytate-containing substrate and allowing the enzymes in the seeds toincrease the rate of reaction. By directly adding the seeds to aphytate-containing substrate, the invention provides a solution to theexpensive and problematic process of extracting and purifying theenzyme. In a particular—but by no means limiting—exemplification, thepresent invention also provides methods of treatment whereby an organismlacking a sufficient supply of an enzyme is administered the enzyme inthe form of seeds containing enhanced amounts of the enzyme. In apreferred embodiment, the timing of the administration of the enzyme toan organism is coordinated with the consumption of a phytate-containingfoodstuff.

The expression of phytase in plants can be achieved by a variety ofmeans. Specifically, for example, technologies are available fortransforming a large number of plant species, including dicotyledonousspecies (e.g. tobacco, potato, tomato, Petunia, Brassica). Additionally,for example, strategies for the expression of foreign genes in plantsare available. Additionally still, regulatory sequences from plant geneshave been identified that are serviceable for the construction ofchimeric genes that can be functionally expressed in plants and in plantcells (e.g. Klee et al., 1987; Clark et al., 1990; Smith et al., 1990).

The introduction of gene constructs into plants can be achieved usingseveral technologies including transformation with Agrobacteriumtumefaciens or Agrobacterium rhizogenes. Non-limiting examples of planttissues that can be transformed thusly include protoplasts, microsporesor pollen, and explants such as leaves, stems, roots, hypocotyls, andcotyls. Furthermore, DNA can be introduced directly into protoplasts andplant cells or tissues by microinjection, electroporation, particlebombardment, and direct DNA uptake.

Proteins may be produced in plants by a variety of expression systems.For instance, the use of a constitutive promoter such as the 35Spromoter of Cauliflower Mosaic Virus (Guilley et al., 1982) isserviceable for the accumulation of the expressed protein in virtuallyall organs of the transgenic plant. Alternatively, the use of promotersthat are highly tissue-specific and/or stage-specific are serviceablefor this invention (Higgins, 1984; Shotwell, 1989) in order to biasexpression towards desired tissues and/or towards a desired stage ofdevelopment. Further details relevant to the expression in plants of thephytase molecules of the instant invention are disclosed, for example,in U.S. Pat. No. 5,770,413 (Van Ooijen et al.) and U.S. Pat. No.5,593,963 (Van Ooijen et al.), although these references do not teachthe inventive molecules of the instant application and instead teach theuse of fungal phytases.

In sum, it is relevant to this invention that a variety of means can beused to achieve the recombinant expression of phytase in a transgenicplant or plant part. Such a transgenic plants and plant parts areserviceable as sources of recombinantly expressed phytase, which can beadded directly to phytate-containing sources. Alternatively, therecombinant plant-expressed phytase can be extracted away from the plantsource and, if desired, purified prior to contacting the phytasesubstrate.

Within the context of the present invention, plants to be selectedinclude, but are not limited to crops producing edible flowers such ascauliflower (Brassica oleracea), artichoke (Cynara scolymus), fruitssuch as apple (Malus, e.g. domesticus), banana (Musa, e.g. acuminata),berries (such as the currant, Ribes, e.g. rubrum), cherries (such as thesweet cherry, Prunus, e.g. avium), cucumber (Cucumis, e.g. sativus),grape (Vitis, e.g. vinifera), lemon (Citrus limon), melon (Cucumismelo), nuts (such as the walnut, Juglans, e.g. regia; peanut, Arachishypogeae), orange (Citrus, e.g. maxima), peach (Prunus, e.g. persica),pear (Pyra, e.g. communis), plum (Prunus, e.g. domestica), strawberry(Fragaria, e.g. moschata), tomato (Lycopersicon, e.g. esculentum),leafs, such as alfalfa (Medicago, e.g. sativa), cabbages (e.g. Brassicaoleracea), endive (Cichoreum, e.g. endivia), leek (Allium, e.g. porrum),lettuce (Lactuca, e.g. sativa), spinach (Spinacia, e.g. oleraceae),tobacco (Nicotiana, e.g. tabacum), roots, such as arrowroot (Maranta,e.g. arundinacea), beet (Beta, e.g vulgaris), carrot (Daucus, e.g.carota), cassaya (Manihot, e.g. esculenta), turnip (Brassica, e.g.rapa), radish (Raphanus, e.g. sativus), yam (Dioscorea, e.g. esculenta),sweet potato (Ipomoea batatas) and seeds, such as bean (Phaseolus, e.g.vulgaris), pea (Pisum, e.g. sativum), soybean (Glycin, e.g. max), wheat(Triticum, e.g. aestivum), barley (Hordeum, e.g. vulgare), corn (Zea,e.g. mays), rice (Oryza, e.g. sativa), rapeseed (Brassica napus), millet(Panicum L.), sunflower (Helianthus annus), oats (Avena sativa), tubers,such as kohlrabi (Brassica, e.g. oleraceae), potato (Solanum, e.g.tuberosum) and the like.

It is understood that additional plant as well as non-plant expressionsystems can be used within the context of this invention. The choice ofthe plant species is primarily determined by the intended use of theplant or parts thereof and the amenability of the plant species totransformation.

Several techniques are available for the introduction of the expressionconstruct containing the phytase-encoding DNA sequence into the targetplants. Such techniques include but are not limited to transformation ofprotoplasts using the calcium/polyethylene glycol method,electroporation and microinjection or (coated) particle bombardment(Potrykus, 1990). In addition to these so-called direct DNAtransformation methods, transformation systems involving vectors arewidely available, such as viral vectors (e.g. from the CauliflowerMosaic Cirus (CaMV) and bacterial vectors (e.g. from the genusAgrobacterium) (Potrykus, 1990). After selection and/or screening, theprotoplasts, cells or plant parts that have been transformed can beregenerated into whole plants, using methods known in the art (Horsch etal., 1985). The choice of the transformation and/or regenerationtechniques is not critical for this invention.

For dicots, a preferred embodiment of the present invention uses theprinciple of the binary vector system (Hoekema et al., 1983; EP 0120516Schilperoort et al.) in which Agrobacterium strains are used whichcontain a vir plasmid with the virulence genes and a compatible plasmidcontaining the gene construct to be transferred. This vector canreplicate in both E. coli and in Agrobacterium, and is derived from thebinary vector Bin19 (Bevan, 1984) which is altered in details that arenot relevant for this invention. The binary vectors as used in thisexample contain between the left- and right-border sequences of theT-DNA, an identical NPTII-gene coding for kanamycin resistance (Bevan,1984) and a multiple cloning site to clone in the required geneconstructs.

The transformation and regeneration of monocotyledonous crops is not astandard procedure. However, recent scientific progress shows that inprinciple monocots are amenable to transformation and that fertiletransgenic plants can be regenerated from transformed cells. Thedevelopment of reproducible tissue culture systems for these crops,together with the powerfull methods for introduction of genetic materialinto plant cells has facilitated transformation. Presently the methodsof choice for transformation of monocots are microprojectile bombardmentof explants or suspension cells, and direct DNA uptake orelectroporation of protoplasts. For example, transgenic rice plants havebeen successfully obtained using the bacterial hph gene, encodinghygromycin resistance, as a selection marker. The gene was introduced byelectroporation (Shimamoto et al., 1993). Transgenic maize plants havebeen obtained by introducing the Streptomyces hygroscopicus bar gene,which encodes phosphinothricin acetyltransferase (an enzyme whichinactivates the herbicide phosphinothricin), into embryogenic cells of amaize suspension culture by microparticle bombardment (Gordon-Kamm etal., 1990). The introduction of genetic material into aleuroneprotoplasts of other monocot crops such as wheat and barley has beenreported (Lee et al., 1989). Wheat plants have been regenerated fromembryogenic suspension culture by selecting only the aged compact andnodular embryogenic callus tissues for the establishment of theembryogenic suspension cultures (Vasil et al., 1972: Vasil et al.,1974). The combination with transformation systems for these cropsenables the application of the present invention to monocots. Thesemethods may also be applied for the transformation and regeneration ofdicots.

Expression of the phytase construct involves such details astranscription of the gene by plant polymerases, translation of mRNA,etc. that are known to persons skilled in the art of recombinant DNAtechniques. Only details relevant for the proper understanding of thisinvention are discussed below. Regulatory sequences which are known orare found to cause expression of phytase may be used in the presentinvention. The choice of the regulatory sequences used depends on thetarget crop and/or target organ of interest. Such regulatory sequencesmay be obtained from plants or plant viruses, or may be chemicallysynthesized. Such regulatory sequences are promoters active in directingtranscription in plants, either constitutively or stage and/or tissuespecific, depending on the use of the plant or parts thereof. Thesepromoters include, but are not limited to promoters showing constitutiveexpression, such as the ³⁵S promoter of Cauliflower Mosaic Virus (CaMV)(Guilley et al., 1982), those for leaf-specific expression, such as thepromoter of the ribulose bisphosphate carboxylase small subunit gene(Coruzzi et al., 1984), those for root-specific expression, such as thepromoter from the glutamin synthase gene (Tingey et al., 1987), thosefor seed-specific expression, such as the cruciferin A promoter fromBrassica napus (Ryan et al., 1989), those for tuber-specific expression,such as the class-I patatin promoter from potato (Koster-Topfer et al.,1989; Wenzler et al., 1989) or those for fruit-specific expression, suchas the polygalacturonase (PG) promoter from tomato (Bird et al., 1988).

Other regulatory sequences such as terminator sequences andpolyadenylation signals include any such sequence functioning as such inplants, the choice of which is within the level of the skilled artisan.An example of such sequences is the 3′ flanking region of the nopalinesynthase (nos) gene of Agrobacterium tumefaciens (Bevan, supra). Theregulatory sequences may also include enhancer sequences, such as foundin the ³⁵S promoter of CaMV, and mRNA stabilizing sequences such as theleader sequence of Alfalfa Mosaic Cirus (AlMV) RNA4 (Brederode et al.,1980) or any other sequences functioning in a like manner.

The phytase should be expressed in an environment that allows forstability of the expressed protein. The choice of cellular compartments,such as cytosol, endoplasmic reticulum, vacuole, protein body orperiplasmic space can be used in the present invention to create such astable environment, depending on the biophysical parameters of thephytase. Such parameters include, but are not limited to pH-optimum,sensitivity to proteases or sensitivity to the molarity of the preferredcompartment.

To obtain expression in the cytoplasm of the cell, the expressed enzymeshould not contain a secretory signal peptide or any other targetsequence. For expression in chloroplasts and mitochondria the expressedenzyme should contain specific so-called transit peptide for import intothese organelles. Targeting sequences that can be attached to the enzymeof interest in order to achieve this are known (Smeekens et al., 1990;van den Broeck et al., 1985; Wolter et al., 1988). If the activity ofthe enzyme is desired in the vacuoles a secretory signal peptide has tobe present, as well as a specific targeting sequence that directs theenzyme to these vacuoles (Tague et al., 1990). The same is true for theprotein bodies in seeds. The DNA sequence encoding the enzyme ofinterest should be modified in such a way that the enzyme can exert itsaction at the desired location in the cell.

To achieve extracellular expression of the phytase, the expressionconstruct of the present invention utilizes a secretory signal sequence.Although signal sequences which are homologous (native) to the planthost species are preferred, heterologous signal sequences, i.e. thoseoriginating from other plant species or of microbial origin, may be usedas well. Such signal sequences are known to those skilled in the art.Appropriate signal sequences which may be used within the context of thepresent invention are disclosed in Blobel et al., 1979; Von Heijne,1986; Garcia et al., 1987; Sijmons et al., 1990; Ng et al., 1994; andPowers et al., 1996).

All parts of the relevant DNA constructs (promoters, regulatory-,secretory-, stabilizing-, targeting-, or termination sequences) of thepresent invention may be modified, if desired, to affect their controlcharacteristics using methods known to those skilled in the art. It ispointed out that plants containing phytase obtained via the presentinvention may be used to obtain plants or plant organs with yet higherphytase levels. For example, it may be possible to obtain such plants orplant organs by the use of somoclonal variation techniques or by crossbreeding techniques. Such techniques are well known to those skilled inthe art.

In one embodiment, the instant invention provides a method (and productsthereof) of achieving a highly efficient overexpression system forphytase and other molecules. In one aspect, the instant inventionprovides a method (and products thereof) of achieving a highly efficientoverexpression system for phytase and pH 2.5 acid phosphatase inTrichoderma. This system results in enzyme compositions that haveparticular utility in the animal feed industry. Additional detailsregarding this approach are in the public literature and/or are known tothe skilled artisan. In a particular non-limiting exemplification, suchpublicly available literature includes EP 0659215 (WO 9403612 A1)(Nevalainen et al.), although these references do not teach theinventive molecules of the instant application.

In one embodiment, the instant invention provides a method (and productsthereof) of producing stabilized aqueous liquid formulations havingphytase activity that exhibit increased resistance to heat inactivationof the enzyme activity and which retain their phytase activity duringprolonged periods of storage. The liquid formulations are stabilized bymeans of the addition of urea and/or a polyol such as sorbitol andglycerol as stabilizing agent. Also provided are feed preparations formonogastric animals and methods for the production thereof that resultfrom the use of such stabilized aqueous liquid formulations. Additionaldetails regarding this approach are in the public literature and/or areknown to the skilled artisan. In a particular non-limitingexemplification, such publicly available literature includes EP 0626010(WO 9316175 A1) (Barendse et al.), although references in the publiclyavailable literature do not teach the inventive molecules of the instantapplication.

In one embodiment, the instant invention provides a method ofhydrolyzing phytate comprised of contacting the phytate with one or moreof the novel phytase molecules disclosed herein. Accordingly, theinvention provides a method for catalyzing the hydrolysis of phytate toinositol and free phosphate with release of minerals from the phyticacid complex. The method includes contacting a phytate substrate with adegrading effective amount of an enzyme of the invention, such as theenzyme shown in SEQ ID NO:2. The term “degrading effective” amountrefers to the amount of enzyme which is required to degrade at least 50%of the phytate, as compared to phytate not contacted with the enzyme.Preferably, at least 80% of the phytate is degraded.

In another embodiment, the invention provides a method for hydrolyzingphospho-mono-ester bonds in phytate. The method includes administering(e.g., to an individual, e.g., a human, or an animal) an effectiveamount of a phytase of the invention (e.g., a phytase having a sequenceidentity of at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98%, 98.5%,99%, 99.5%, or more, or complete (100%) sequence identity (i.e.,homology) to SEQ ID NO:2 (a phytase polypeptide); SEQ ID NO:10 (aphytase polypeptide); a polypeptide having sequence as set forth in SEQID NO:8 and having at least one, or all, of the amino acid modificationsW68E, Q84W, A95P, K97C, S168E, R181Y, N226C, Y277D, wherein thepolypeptide has phytase activity) or other phytases, for example, the E.coli appA “wild type” phytase-encoding SEQ ID NO:7, or, a polypeptidesequence of SEQ ID N 0:2 or the E. coli appA “wild type” phytase SEQ IDNO:8, to yield inositol and free phosphate. An “effective” amount refersto the amount of enzyme which is required to hydrolyze at least 50% ofthe phospho-mono-ester bonds, as compared to phytate not contacted withthe enzyme.

In a particular aspect, when desired, the phytase molecules may be usedin combination with other reagents, such as other catalysts; in order toeffect chemical changes (e.g. hydrolysis) in the phytate moleculesand/or in other molecules of the substrate source(s). According to thisaspect, the phytase molecules and the additional reagent(s) will notinhibit each other, or, the phytase molecules and the additionalreagent(s) will have an overall additive effect, or, the phytasemolecules and the additional reagent(s) will have an overall synergisticeffect.

Relevant sources of the substrate phytate molecules include foodstuffs,potential foodstuffs, byproducts of foodstuffs (both in vitro byproductsand in vivo byproducts, e.g. ex vivo reaction products and animalexcremental products), precursors of foodstuffs, and any other materialsource of phytate.

In a non-limiting apsect, the recombinant phytase can be consumed byorganisms and retains activity upon consumption. In anotherexemplification, transgenic approches can be used to achieve expressionof the recombinant phytase—in one aspect, in a controlled fashion(methods are available for controlling expression of transgenicmolecules in time-specific and tissue specific manners).

In a particular exemplification, the phytase activity in the sourcematerial (e.g. a transgenic plant source or a recombinant prokaryotichost) may be increased upon consumption; this increase in activity mayoccur, for example, upon conversion of a precursor phytase molecule inpro-form to a significantly more active enzyme in a more mature form,where said conversion may result, for example, from the injestion anddigestion of the phytase source. Hydrolysis of the phytate substrate mayoccur at any time upon the contacting of the phytase with the phytate;for example, this may occur before injestion or after injestion or bothbefore and after injestion of either the substrate or the enzyme orboth. It is additionally appreciated that the phytate substrate may becontacted with—in addition to the phytase—one or more additionalreagents, such as another enzyme, which may be also be applied eitherdirectly or after purification from its source material.

It is appreciated that the phytase source material(s) can be contacteddirectly with the phytate source material(s); e.g. upon in vitro or invivo grinding or chewing of either or both the phytase source(s) and thephytate source(s). Alternatively the phytase enzyme may be purified awayfrom source material(s), or the phytate substrate may be purified awayfrom source material(s), or both the phytase enzyme and the phytatesubstrate may be purified away from source material(s) prior to thecontacing of the phytase enzyme with the phytate substrate. It isappreciated that a combination of purified and unpurifiedreagents—including enzyme(s) or substrates(s) or both—may be used.

It is appreciated that more than one source material may be used as asource of phytase activity. This is serviceable as one way to achieve atimed release of reagent(s) from source material(s), where release fromdifferent reagents from their source materials occur differentially, forexample as injested source materials are digested in vivo or as sourcematerials are processed in in vitro applications. The use of more thanone source material of phytase activity is also serviceable to obtainphytase activities under a range of conditions and fluctuations thereof,that may be encountered—such as a range of pH values, temperatures,salinities, and time intervals—for example during different processingsteps of an application. The use of different source materials is alsoserviceable in order to obtain different reagents, as exemplified by oneor more forms or isomers of phytase and/or phytate &/or other materials.

It is appreciated that a single source material, such a trangenic plantspecies (or plant parts thereof), may be a source material of bothphytase and phytate; and that enzymes and substrates may bedifferentially compartmentalized within said single source—e.g. secretedvs.-non-secreted, differentially expressed &/or having differentialabundances in different plant parts or organs or tissues or insubcellular compartments within the same plant part or organ or tissue.Purification of the phytase molecules contained therein may compriseisolating and/or further processing of one or more desirable plant partsor organs or tissues or subcellular compartments.

In a particular aspect, this invention provides a method of catalyzingin vivo and/or in vitro reactions using seeds containing enhancedamounts of enzymes. The method comprises adding transgenic, non-wildtype seeds, preferably in a ground form, to a reaction mixture andallowing the enzymes in the seeds to increase the rate of reaction. Bydirectly adding the seeds to the reaction mixture the method provides asolution to the more expensive and cumbersome process of extracting andpurifying the enzyme. Methods of treatment are also provided whereby anorganism lacking a sufficient supply of an enzyme is administered theenzyme in the form of seeds from one or more plant species, preferablytransgenic plant species, containing enhanced amounts of the enzyme.Additional details regarding this approach are in the public literatureand/or are known to the skilled artisan. In a particular non-limitingexemplification, such publicly available literature includes U.S. Pat.No. 5,543,576 (Van Ooijen et al.) and U.S. Pat. No. 5,714,474 (VanOoijen et al.), although these reference do not teach the inventivemolecules of the instant application and instead teach the use of fungalphytases.

In one aspect, the instant phytase molecules are serviceable forgenerating recombinant digestive system life forms (or microbes orflora) and for the administration of said recombinant digestive systemlife forms to animals. Administration may be optionally performed aloneor in combination with other enzymes &/or with other life forms that canprovide enzymatic activity in a digestive system, where said otherenzymes and said life forms may be recombinant or otherwise. Forexample, administration may be performed in combination with xylanolyticbacteria

In a non-limiting aspect, the present invention provides a method forsteeping corn or sorghum kernels in warm water containing sulfur dioxidein the presence of an enzyme preparation comprising one or morephytin-degrading enzymes, preferably in such an amount that the phytinpresent in the corn or sorghum is substantially degraded. The enzymepreparation may comprise phytase and/or acid phosphatase and optionallyother plant material degrading enzymes. The steeping time may be 12 to18 hours. The steeping may be interrupted by an intermediate millingstep, reducing the steeping time. In a preferred embodiment, corn orsorghum kernels are steeped in warm water containing sulfur dioxide inthe presence of an enzyme preparation including one or morephytin-degrading enzymes, such as phytase and acid phosphatases, toeliminate or greatly reduce phytic acid and the salts of phytic acid.Additional details regarding this approach are in the public literatureand/or are known to the skilled artisan. In a particular non-limitingexemplification, such publicly available literature includes U.S. Pat.No. 4,914,029 (Caransa et al.) and EP 0321004 (Vaara et al.), althoughthese references do not teach the inventive molecules of the instantapplication.

In a non-lirniting aspect, the present invention provides a method toobtain a bread, dough having desirable physical properties such asnon-tackiness and elasticity and a bread product of superior qualitysuch as a specific volume comprising adding phytase molecules to thebread dough. In a preferred embodiment, phytase molecules of the instantinvention are added to a working bread dough preparation that issubsequently formed and baked. Additional details regarding thisapproach are in the public literature and/or are known to the skilledartisan. In a particular non-limiting exemplification, such publiclyavailable literature includes JP 03076529 (Hara et al.), although thisreference does not teach the inventive phytase molecules of the instantapplication.

In a non-limiting aspect, the present invention provides a method toproduce improved soybean foodstuffs. Soybeans are combined with phytasemolecules of the instant invention to remove phytic acid from thesoybeans, thus producing soybean foodstuffs that are improved in theirsupply of trace nutrients essential for consuming organisms and in itsdigestibility of proteins. In a preferred embodiment, in the productionof soybean milk, phytase molecules of the instant invention are added toor brought into contact with soybeans in order to reduce the phytic acidcontent. In a non-limiting exemplification, the application process canbe accelerated by agitating the soybean milk together with the enzymeunder heating or by a conducting a mixing-type reaction in an agitationcontainer using an immobilized enzyme. Additional details regarding thisapproach are in the public literature and/or are known to the skilledartisan. In a particular non-limiting exemplification, such publiclyavailable literature includes JP 59166049 (Kamikubo et al.), althoughthis reference does not teach the inventive molecules of the instantapplication.

In one aspect, the instant invention provides a method of producing anadmixture product for drinking water or animal feed in fluid form, andwhich comprises using mineral mixtures and vitamin mixtures, and alsonovel phytase molecules of the instant invention. In a preferredembodiment, there is achieved a correctly dosed and composed mixture ofnecessary nutrients for the consuming organism without any risk ofprecipitation and destruction of important minerals/vitamins, while atthe same time optimum utilization is made of the phytin-bound phosphatein the feed. Additional details regarding this approach are in thepublic literature and/or are known to the skilled artisan. In aparticular non-limiting exemplification, such publicly availableliterature includes EP 0772978 (Bendixen et al.), although thisreference does not teach the inventive molecules of the instantapplication.

It is appreciated that the phytase molecules of the instant inventionmay also be used to produce other alcoholic and non-alcoholic drinkablefoodstuffs (or drinks) based on the use of molds &/or on grains &/or onother plants. These drinkable foodstuffs include liquors, wines, mixedalcoholic drinks (e.g. wine coolers, other alcoholic coffees such asIrish coffees, etc.), beers, near-beers, juices, extracts, homogenates,and purees. In a preferred exemplification, the instantly disclosedphytase molecules are used to generate transgenic versions of molds &/orgrains &/or other plants serviceable for the production of suchdrinkable foodstuffs. In another preferred exemplification, theinstantly disclosed phytase molecules are used as additional ingredientsin the manufacturing process &/or in the final content of such drinkablefoodstuffs. Additional details regarding this approach are in the publicliterature and/or are known to the skilled artisan. However—due to thenovelty of the instant invention—references in the publicly availableliterature do not teach the inventive molecules instantly disclosed.

In another non-limiting exemplification, the present invention providesa means to obtain refined sake having a reduced amount of phytin and anincreased content of inositol. Such a sake may have—through direct &/orpsychogenic effects—a preventive action on hepatic disease,arteriosclerosis, and other diseases. In a preferred embodiment, a sakeis produced from rice Koji by multiplying a rice Koji mold having highphytase activity as a raw material. It is appreciated that the phytasemolecules of the instant invention may be used to produce a serviceablemold with enhanced activity (preferably a transgenic mold) &/or addedexogenously to augment the effects of a Koji mold. The strain is addedto boiled rice and Koji is produced by a conventional procedure. In apreferred exemplification, the prepared Koji is used, the whole rice isprepared at two stages and Sake is produced at constant Sake temperatureof 15° C. to give the objective refined Sake having a reduced amount ofphytin and an increased amount of inositol. Additional details regardingthis approach are in the public literature and/or are known to theskilled artisan. In a particular non-limiting exemplification, suchpublicly available literature includes JP 06153896 (Soga et al.) and JP06070749 (Soga et al.), although these references do not teach theinventive molecules of the instant application

In a non-limiting aspect, the present invention provides a method toobtain an absorbefacient capable of promoting the absorption of mineralsincluding ingested calcium without being digested by gastric juices orintestinal juices at a low cost. In a preferred embodiment, said mineralabsorbefacient contains a partial hydrolysate of phytic acid as anactive ingredient. Preferably, a partial hydrolyzate of the phytic acidis produced by hydrolyzing the phytic acid or its salts using novelphytase molecules of the instant invention. The treatment with saidphytase molecules may occur either alone &/or in a combination treatment(to inhibit or to augment the final effect), and is followed byinhibiting the hydrolysis within a range so as not to liberate all thephosphate radicals. Additional details regarding this approach are inthe public literature and/or are known to the skilled artisan. In aparticular non-limiting exemplification, such publicly availableliterature includes JP 04270296 (Hoshino), although reference in thepublicly available literature do not teach the inventive molecules ofthe instant application.

In a non-limiting aspect, the present invention provides a method (andproducts therefrom) to produce an enzyme composition having an additiveor preferably a synergistic phytate hydrolyzing activity; saidcomposition comprises novel phytase molecules of the instant inventionand one or more additional reagents to achieve a composition that isserviceable for a combination treatment. In a preferred embodiment, thecombination treatment of the present invention is achieved with the useof at least two phytases of different position specificity, i.e. anycombinations of 1-, 2-, 3-, 4-, 5-, and 6-phytases. By combiningphytases of different position specificity an additive or synergisticeffect is obtained. Compositions such as food and feed or food and feedadditives comprising such phytases in combination are also included inthis invention as are processes for their preparation. Additionaldetails regarding this approach are in the public literature and/or areknown to the skilled artisan. In a particular non-limitingexemplification, such publicly available literature includes WO9 830681(Ohmann et al.), although references in the publicly availableliterature do not teach the use of the inventive molecules of theinstant application.

In one aspect, the combination treatment of the present invention isachieved with the use of an acid phosphatase having phytate hydrolyzingactivity at a pH of 2.5, in a low ratio corresponding to a pH 2.5:5.0activity profile of from about 0.1:1.0 to 10:1, preferably of from about0.5:1.0 to 5:1, more preferably still of from about 0.8:1.0 to 3:1, andmore preferably still of from about 0.8:1.0 to 2:1. Said enzymecomposition can displays a higher synergetic phytate hydrolyzingefficiency through thermal treatment. Said enzyme composition isserviceable in the treatment of foodstuffs (drinkable and solid food,feed and fodder products) to improve phytate hydrolysis. Additionaldetails regarding this approach are in the public literature and/or areknown to the skilled artisan. In a particular non-limitingexemplification, such publicly available literature includes U.S. Pat.No. 5,554,399 (Vanderbeke et al.) and U.S. Pat. No. 5,443,979(Vanderbeke et al.), although these reference do not teach the use ofthe inventive molecules of the instant application, but rather teach theuse of fungal (in particular Aspegillus) phytases.

In a non-limiting aspect, the present invention provides a method (andproducts therefrom) to produce compositions comprised of the instantnovel phytate-acting enzyme in combination with one or more additionalenzymes that act on polysaccharides. Such polysaccharides can beselected from the group consisting of arabinans, fructans, fucans,galactans, galacturonans, glucans, mannans, xylans, levan, fucoidan,carrageenan, galactocarolose, pectin, pectic acid, amylose, pullulan,glycogen, amylopectin, cellulose, carboxylmethylcellulose,hydroxypropylmethylcellulose, dextran, pustulan, chitin, agarose,keratan, chondroitin, dermatan, hyaluronic acid, alginic acid, andpolysaccharides containing at least one aldose, ketose, acid or amineselected from the group consisting of erythrose, threose, ribose,arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose,idose, galactose, talose, erythrulose, ribulose, xylulose, psicose,fructose, sorbose, tagatose, glucuronic acid, gluconic acid, glucaricacid, galacturonic acid, mannuronic acid, glucosamine, galactosamine andneuraminic acid

In a particular aspect, the present invention provides a method (andproducts therefrom) to produce composition having a synergistic phytatehydrolyzing activity comprising one or more novel phytase molecules ofthe instant invention, a cellulase (in one aspect including but notexclusively a xylanase), optionally a protease, and optionally one ormore additonal reagents. In preferred embodiments, such combinationtreatments are serviceable in the treatment of foodstuffs, woodproducts, such as paper products, and as cleansing solutions and solids.

In one non-limiting exemplification, the instant phytase molecules areserviceable in combination with cellulosome components. It is known thatcellulases of many cellulolytic bacteria are organized into discretemultienzyme complexes, called cellulosomes. The multiple subunits ofcellulosomes are composed of numerous functional domains, which interactwith each other and with the cellulosic substrate. One of these subunitscomprises a distinctive new class of noncatalytic scaffoldingpolypeptide, which selectively integrates the various cellulase andxylanase subunits into the cohesive complex. Intelligent application ofcellulosome hybrids and chimeric constructs of cellulosomal domainsshould enable better use of cellulosic biomass and may offer a widerange of novel applications in research, medicine and industry.

In another non-limiting exemplification, the instant phytase moleculesare serviceable—either alone or in combination treatments—in areas ofbiopulping and biobleaching where a reduction in the use ofenvironmentally harmful chemicals traditionally used in the pulp andpaper industry is desired. Waste water treatment represents another vastapplication area where biological enzymes have been shown to beeffective not only in colour removal but also in the bioconversion ofpotentially noxious substances into useful bioproducts.

In another non-limiting exemplification, the instant phytase moleculesare serviceable for generating life forms that can provide at least oneenzymatic activity—either alone or in combination treatments—in thetreatment of digestive systems of organisms. Particularly relevantorganisms to be treated include non-ruminant organisms. Specifically, itis appreciated that this approach may be performed alone or incombination with other biological molecules (for example, xylanases) togenerate a recombinant host that expresses a plurality of biologicalmolecules. It is also appreciated that the administration of the instantphytase molecules &/or recombinant hosts expressing the instant phytasemolecules may be performed either alone or in combination with otherbiological molecules, &/or life forms that can provide enzymaticactivities in a digestive system—where said other enzymes and said lifeforms may be may recombinant or otherwise. For example, administrationmay be performed in combination with xylanolytic bacteria

For example, in addition to phytate, many organisms are also unable toadequately digest hemicelluloses. Hemicelluloses or xylans are majorcomponents (35%) of plant materials. For ruminant animals, about 50% ofthe dietary xylans are degraded, but only small amounts of xylans aredegraded in the lower gut of nonruminant animals and humans. In therumen, the major xylanolytic species are Butyrivibrio fibrisolvens andBacteroides rumiincola. In the human colon, Bacteroides ovatus andBacteroides fragilis subspecies “a” are major xylanolytic bacteria.Xylans are chemically complex, and their degradation requires multipleenzymes. Expression of these enzymes by gut bacteria varies greatlyamong species. Butyrivibrio fibrisolvens makes extracellular xylanasesbut Bacteroides species have cell-bound xylanase activity. Biochemicalcharacterization of xylanolytic enzymes from gut bacteria has not beendone completely. A xylosidase gene has been cloned from B. fibrosolvens113. The data from DNA hybridizations using a xylanase gene cloned fromB. fibrisolvens 49 indicate this gene may be present in other B.fibrisolvens strains. A cloned xylanase from Bact. ruminicola wastransferred to and highly expressed in Bact. fragilis and Bact.uniformis. Arabinosidase and xylosidase genes from Bact. ovatus havebeen cloned and both activities appear to be catalyzed by a single,bifunctional, novel enzyme.

Accordingly, it is appreciated that the present phytase molecules areserviceable for 1) transferring into a suitable host (such as Bact.fragilis or Bact. uniformis); 2) achieving adequate expression in aresultant recombinant host; and 3) administering said recombinant hostto organisms to improve the ability of the treated organisms to degradephytate. Continued research in genetic and biochemical areas willprovide knowledge and insights for manipulation of digestion at the gutlevel and improved understanding of colonic fiber digestion.

Additional details regarding this approach are in the public literatureand/or are known to the skilled artisan. In a particular non-limitingexemplification, such publicly available literature includes U.S. Pat.No. 5,624,678 (Bedford et al.), U.S. Pat. No. 5,683,911 (Bodie et al.),U.S. Pat. No. 5,720,971 (Beauchemin et al.), U.S. Pat. No. 5,759,840(Sung et al.), U.S. Pat. No. 5,770,012 (Cooper), U.S. Pat. No. 5,786,316(Baeck et al.), U.S. Pat. No. 5,817,500 (Hansen et al.), and journalarticles (Jeffries, 1996; Prade, 1996; Bayer et al., 1994; Duarte etal., 1994; Hespell & Whitehead, 1990; Wong et al., 1988), although thesereference do not teach the inventive phytase molecules of the instantapplication, nor do they all teach the addition of phytase molecules inthe production of foodstuffs, wood products, such as paper products, andas cleansing solutions and solids. In contrast, the instant inventionteaches that phytase molecules—preferably the inventive phytasemolecules of the instant application—may be added to the reagent(s)disclosed in order to obtain preparations having an additional phytaseactivity. Preferably, said reagent(s) and the additional phytasemolecules and will not inhibit each other, more preferably saidreagent(s) and the additional phytase molecules will have an overalladditive effect, and more preferably still said reagent(s) and theadditional phytase molecules will have an overall synergistic effect.

In a non-limiting aspect, the present invention provides a method (andproducts therefrom) for enhancement of phytate phosphorus utilizationand treatment and prevention of tibial dyschondroplasia in animals,particularly poultry, by administering to animals a feed compositioncontaining a hydroxylated vitamin D₃ derivative. The vitamin D₃derivative can be administered to animals in feed containing reducedlevels of calcium and phosphorus for enhancement of phytate phosphorusutilization. Accordingly, the vitamin D₃ derivative can be administeredin combination with novel phytase molecules of the instant invention forfurther enhancement of phytate phosphorus utilization. Additionaldetails regarding this approach are in the public literature and/or areknown to the skilled artisan. In a particular non-limitingexemplification, such publicly available literature includes U.S. Pat.No. 5,516,525 (Edwards et al.) and U.S. Pat. No. 5,366,736 (Edwards etal.), U.S. Pat. No. 5,316,770 (Edwards et al.) although these referencesdo not teach the inventive molecules of the instant application.

In a non-limiting aspect, the present invention provides a method (andproducts therefrom) to obtain foodstuff that 1) comprises phytin that iseasily absorbed and utilized in a form of inositol in a body of anorganism; 2) that is capable of reducing phosphorus in excrementarymatter; and 3) that is accordingly useful for improving environmentalpollution. Said foodstuff is comprised of an admixture of aphytin-containing grain, a lactic acid-producing microorganism, and anovel phytase molecule of the instant invention. In a preferredembodiment, said foodstuff is produced by compounding aphytin-containing grain (preferably, e.g. rice bran) with an effectivemicrobial group to be added having an acidophilic property, producinglactic acid, without producing butyric acid, free from pathogenicity,and a phytase. Examples of an effective microbial group to be addedinclude e.g. Streptomyces sp. (ATCC 3004) belonging to the group ofactinomyces and Lactobacillus sp. (IFO 3070) belonging to the group oflactobacilli. Further, a preferable amount of an effective microbialgroup to be added is 0.2 wt. % in terms of bacterial body weight basedon a grain material. Furthermore, the amount of the addition of thephytase to be added can be 1-2 wt. % based on the phytin in the grainmaterial. Additional details regarding this approach are in the publicliterature and/or are known to the skilled artisan. In a particularnon-limiting exemplification, such publicly available literatureincludes JP 08205785 (Akahori et al.), although references in thepublicly available literature do not teach the inventive molecules ofthe instant application.

In a non-limiting aspect, the present invention provides a method forimproving the solubility of vegetable proteins. More specifically, theinvention relates to methods for the solubilization of proteins invegetable protein sources, which methods comprise treating the vegetableprotein source with an effective amount of one or more phytaseenzymes—including phytase molecules of the instant invention—andtreating the vegetable protein source with an effective amount of one ormore proteolytic enzymes. In another aspect, the invention providesanimal feed additives comprising a phytase and one or more proteolyticenzymes. Additional details regarding this approach are in the publicliterature and/or are known to the skilled artisan. In a particularnon-limiting exemplification, such publicly available literatureincludes EP 0756457 (WO 9528850 A1) (Nielsen and Knap), althoughreferences in the publicly available literature do not teach theinventive molecules of the instant application.

In a non-limiting aspect, the present invention provides a method ofproducing a plant protein preparation comprising dispersing vegetableprotein source materials in water at a pH in the range of 2 to 6 andadmixing phytase molecules of the instant invention therein. The acidicextract containing soluble protein is separated and dried to yield asolid protein of desirable character. One or more proteases can also beused to improve the characteristics of the protein. Additional detailsregarding this approach are in the public literature and/or are known tothe skilled artisan. In a particular non-limiting exemplification, suchpublicly available literature includes U.S. Pat. No. 3,966,971(Morehouse et al.), although references in the publicly availableliterature do not teach the inventive molecules of the instantapplication.

In a non-limiting aspect, the present invention provides a method (andproducts thereof) to activate inert phosphorus in soil and/or compost,to improve the utilization rate of a nitrogen compound, and to suppresspropagation of pathogenic molds by adding three reagents, phytase,saponin and chitosan, to the compost. In a non-limiting embodiment themethod can comprise treating the compost by 1) adding phytase-containingmicroorganisms in media—preferably recombinant hosts that overexpressthe novel phytase molecules of the instant invention—e.g. at 100 mlmedia/100 kg wet compost; 2) alternatively also adding aphytase-containing plant source—such as wheat bran—e.g. at 0.2 to 1kg/100 kg wet compost; 3) adding a saponin-containing source—such aspeat, mugworts and yucca plants—e.g. at 0.5 to 3.0 g/kg; 4) addingchitosan-containing materials—such as pulverized shells of shrimps,crabs, etc.—e.g. at 100 to 300 g/kg wet compost. In another non-limitingembodiment, recombinant forms the three reagents, phytase, saponin, andchitosan, are used. Additional details regarding this approach are inthe public literature and/or are known to the skilled artisan. In aparticular non-limiting exemplification, such publicly availableliterature includes JP 07277865 (Toya Taisuke), although references inthe publicly available literature do not teach the inventive moleculesof the instant application.

Fragments of the full length gene of the present invention may be usedas a hybridization probe for a cDNA or a genomic library to isolate thefull length DNA and to isolate other DNAs which have a high sequencesimilarity to the gene or similar biological activity. Probes of thistype have at least 10, preferably at least 15, and even more preferablyat least 30 bases and may contain, for example, at least 50 or morebases. The probe may also be used to identify a DNA clone correspondingto a full length transcript and a genomic clone or clones that containthe complete gene including regulatory and promotor regions, exons, andintrons.

The present invention provides methods for identifying nucleic acidmolecules that encode members of the phytase polypeptide family inaddition to SEQ ID NO:1. In these methods, a sample, e.g., a nucleicacid library, such as a cDNA library, that contains a nucleic acidencoding a phytase polypeptide is screened with a phytase-specificprobe, e.g., a phytase-specific nucleic acid probe. Phytase-specificnucleic acid probes are nucleic acid molecules (e.g., moleculescontaining DNA or RNA nucleotides, or combinations or modificationsthereof) that specifically hybridize to nucleic acids encoding phytasepolypeptides, or to complementary sequences thereof. The term“phytase-specific probe,” in the context of this method of invention,refers to probes that bind to nucleic acids encoding phytasepolypeptides, or to complementary sequences thereof, to a detectablygreater extent than to nucleic acids encoding other enzymes, or tocomplementary sequences thereof.

The invention facilitates production of phytase-specific nucleic acidprobes. Methods for obtaining such probes can be designed based on theamino acid sequences shown in FIGS. 1 a and 1 b. The probes, which cancontain at least 12, e.g., at least 15, 25, 35, 50, 100, or 150nucleotides, can be produced using any of several standard methods (see,e.g., Ausubel et al., supra). For example, preferably, the probes aregenerated using PCR amplification methods. In these methods, primers aredesigned that correspond to phytase-conserved sequences (see FIGS. 1 aand 1 b), which can include phytase-specific amino acids, and theresulting PCR product is used as a probe to screen a nucleic acidlibrary, such as a cDNA library.

This invention can be used to isolate nucleic acid sequencessubstantially similar to the isolated nucleic acid molecule encoding anphytase enzyme disclosed in FIGS. 1 a and 1 b (SEQ ID NO:1). Isolatednucleic acid sequences are substantially similar if: (i) they arecapable of hybridizing under stringent conditions, hereinafterdescribed, to SEQ ID NO:1; or (ii) they encode a phytase polypeptide asset forth in SEQ ID NO:2 due to the degeneracy of the genetic code(e.g., degenerate to SEQ ID NO:1).

Degenerate DNA sequences encode the amino acid sequence of SEQ ID NO:2,but have variations in the nucleotide coding sequences. As used herein,“substantially similar” refers to the sequences having similar identityto the sequences of the instant invention. The nucleotide sequences thatare substantially similar can be identified by hybridization or bysequence comparison. Enzyme sequences that are substantially similar canbe identified by one or more of the following: proteolytic digestion,gel electrophoresis and/or microsequencing.

One means for isolating a nucleic acid molecule encoding a phytaseenzyme is to probe a genomic gene library with a natural or artificiallydesigned probe using art recognized procedures (see, e.g., Ausubel etal., supra). It is appreciated to one skilled in the art that SEQ IDNO:1, or fragments thereof (comprising at least 15 contiguousnucleotides), is a particularly useful probe. Other particular usefulprobes for this purpose are hybridizable fragments to the sequences ofSEQ ID NO:1 (i.e., comprising at least 15 contiguous nucleotides).

It is also appreciated that such probes can be and can be labeled withan analytically detectable reagent to facilitate identification of theprobe. Useful reagents include but are not limited to radioactivity,fluorescent dyes or enzymes capable of catalyzing the formation of adetectable product. The probes are thus useful to isolate complementarycopies of DNA from other animal sources or to screen such sources forrelated sequences.

With respect to nucleic acid sequences which hybridize to specificnucleic acid sequences disclosed herein, hybridization may be carriedout under conditions of reduced stringency, medium stringency or evenstringent conditions. As an example of oligonucleotide hybridization, apolymer membrane containing immobilized denatured nucleic acid is firstprehybridized for 30 minutes at 45° C. in a solution consisting of 0.9 MNaCl, 50 mM NaH₂PO₄, pH 7.0, 5.0 mM Na₂EDTA, 0.5% SDS, 10× Denhardt's,and 0.5 mg/mL polyriboadenylic acid. Approximately 2×10⁷ cpm (specificactivity 4-9×10⁸ cpm/ug) of ³²P end-labeled oligenucleotide probe arethen added to the solution. After 12-16 hours of incubation, themembrane is washed for 30 minutes at room temperature in 1× SET (150 mMNaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na₂EDTA) containing 0.5%SDS, followed by a 30 minute wash in fresh 1× SET at Tm-10° C. for theoligo-nucleotide probe. The membrane is then exposed toauto-radiographic film for detection of hybridization signals.

The nucleic acid molecules of the invention can be used as templates instandard methods for production of phytase gene products (e.g., phytaseRNAs and phytase polypeptides). In addition, the nucleic acid moleculesthat encode phytase polypeptides (and fragments thereof) and relatednucleic acids—such as (1) nucleic acids containing sequences that arecomplementary to, or that hybridize to, nucleic acids encoding phytasepolypeptides, or fragments thereof (e.g., fragments containing at least12, 15, 20, or 25 nucleotides); and (2) nucleic acids containingsequences that hybridize to sequences that are complementary to nucleicacids encoding phytase polypeptides, or fragments thereof (e.g.,fragments containing at least 12, 15, 20, or 25 nucleotides)—can be usedin methods focused on their hybridization properties. For example, as isdescribed in further detail herein, such nucleic acid molecules can beused in the following methods: PCR methods for synthesizing phytasenucleic acids, methods for detecting the presence of a phytase nucleicacid in a sample, screening methods for identifying nucleic acidsencoding new phytase family members. Hybridization-based uses includeSouthern-type, Northern-type, RNA protection, and any hybridizationprocedure were a nucleic acid is used as a hybridization partner.

Fragments or portions of the polynucleotides of the present inventionmay be used to synthesize full-length polynucleotides of the presentinvention. Accordingly, fragments or portions of the enzymes of thepresent invention may be employed for producing the correspondingfull-length enzyme by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length enzymes. Sizeseparation of the cleaved fragments is generally performed using 8percent polyacrylamide gel as described in the literature (e.g. byGoeddel et al., 1980).

This invention provides enzymes, as well as fragments, otherderivatives, and analogs thereof, and the corresponding nucleotides foruse in directed evolution. The discovery and use of a plurality oftemplates as disclosed herein may significantly increase the potentialyield of directed evolution in comparison to the directed evolution of asingle template protein. Hence, the need for discovery is based on thepremise that nature provides a wealth of potentially unattainable orunpredictable features in distinct but related members of moleculargroupings, and that the exploitation of these features may greatlyfacilitate directed evolution. Thus, in one aspect, related but distinctmolecules may serve as unique starting templates for the directedevolution of a desired characteristic. In another aspect, they may serveas repositories of structure-function information including, but notlimited to, a variety of consensus motifs. Both utilities help toobviate the logistically impractical task of at once exploring an overlywide range of mutational permutations on any given molecule. Forexample, the full range of mutational permutations on a 100 amino acidprotein includes over 10¹³⁰ possibilities (assuming there are 20 aminoacid possibilities at each position), a number too large for practicalconsideration.

Accordingly, particularly because of logistical and technicalconstraints, it is a desirable approach—in performing “directedevolution”—to discover and to make use of a plurality of relatedstarting templates that have pre-evolved differences. These templatescan then be subjected to a variety of mutagenic manipulations including,by way of non-limiting exemplification, DNA mutagenesis andcombinatorial enzyme development, an approach that is further elaboratedin co-pending U.S. Pat. No. 5,830,696 (Short et al.).

The enzyme activities of the novel molecules generated can then bescreened by a variety of methods including, by way of non-limitingexemplification: a) molecular biopanning; b) recombinant clonescreening; and c) extract screening.

This invention provides enzymes, as well as fragments, otherderivatives, and analogs thereof, and cells expressing them that can beused as an immunogen to produce antibodies thereto. These antibodies canbe, for example, polyclonal or monoclonal antibodies. The presentinvention also includes chimeric, single chain, and humanizedantibodies, as well as Fab fragments, or the product of an Fabexpression library. Various procedures known in the art may be used forthe production of such antibodies and fragments.

Antibodies generated against the enzymes corresponding to a sequence ofthe present invention can be obtained by direct injection of the enzymesinto an animal or by administering the enzymes to an animal, preferablya nonhuman. The antibody so obtained will then bind the enzymes itself.In this manner, even a sequence encoding only a fragment of the enzymescan be used to generate antibodies binding the whole native enzymes.Such antibodies can then be used to isolate the enzyme from cellsexpressing that enzyme.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975),the trioma technique, the human B-cell hybridoma technique (Kozbor etal., 1983), and the EBV-hybridoma technique to produce human monoclonalantibodies (Cole et al., 1985, pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778 Ladner et al.) can be adapted to produce single chainantibodies to immunogenic enzyme products of this invention. Also,transgenic mice may be used to express humanized antibodies toimmunogenic enzyme products of this invention.

Antibodies generated against the enzyme of the present invention may beused in screening for similar enzymes from other organisms and samples.Such screening techniques are known in the art. Antibodies may also beemployed as a probe to screen gene libraries generated from this orother organisms to identify this or cross reactive activities.

Isolation and purification of polypeptides produced in the systemsdescribed above can be carried out using conventional methods,appropriate for the particular system. For example, preparativechromatography and immunological separations employing antibodies, suchas monoclonal or polyclonal antibodies, can be used.

As is mentioned above, antigens that can be used in producingphytase-specific antibodies include phytase polypeptides, e.g., any ofthe phytase shown in Table 3 or polypeptide fragments thereof. Thepolypeptide or peptide used to immunize an animal can be obtained bystandard recombinant, chemical synthetic, or purification methods. As iswell known in the art, in order to increase immunogenicity, an antigencan be conjugated to a carrier protein. Commonly used carriers includekeyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin(BSA), and tetanus toxoid. The coupled peptide is then used to immunizethe animal (e.g., a mouse, a rat, or a rabbit). In addition to suchcarriers, well known adjuvants can be administered with the antigen tofacilitate induction of a strong immune response.

Phytase-specific polyclonal and monoclonal antibodies can be purified,for example, by binding to, and elution from, a matrix containing aphytase polypeptide, e.g., the phytase polypeptide (or fragment thereof)to which the antibodies were raised. Additional methods for antibodypurification and concentration are well known in the art and can bepracticed with the phytase-specific antibodies of the invention (see,e.g., Coligan et al., 1996).

Anti-idiotype antibodies corresponding to phytase-specific antigens arealso included in the invention, and can be produced using standardmethods. These antibodies are raised to phytase-specific antibodies, andthus mimic phytase-specific epitopes.

This invention also includes additonal uses of fragments of the phytasepolypeptides that retain at least one phytase-specific activity orepitope. Phytase activity can be assayed by examining the catalysis ofphytate to inositol and free phosphate. Such fragments can easily beidentified by comparing the sequences of phytases found in FIGS. 1 a and1 b.

In a non-limiting exemplification, a phytase polypeptide fragmentcontaining, e.g., at least 8-10 amino acids can be used as an immunogenin the production of phytase-specific antibodies. The fragment cancontain, for example, an amino acid sequence that is conserved inphytases, and this amino acid sequence can contain amino acids that areconserved in phytases. In another non-limiting exemplification, theabove-described phytase fragments can be used in immunoassays, such asELISAs, to detect the presence of phytase-specific antibodies insamples.

Various methods to make the transgenic animals of the subject inventioncan be employed. Generally speaking, three such methods may be employed.In one such method, an embryo at the pronuclear stage (a “one cellembryo”) is harvested from a female and the transgene is microinjectedinto the embryo, in which case the transgene will be chromosomallyintegrated into both the germ cells and somatic cells of the resultingmature animal. In another such method, embryonic stem cells are isolatedand the transgene is incorporated therein by electroporation, plasmidtransfection or microinjection, followed by reintroduction of the stemcells into the embryo where they colonize and contribute to the germline. Methods for microinjection of mammalian species is described inU.S. Pat. No. 4,873,191. In yet another such method, embryonic cells areinfected with a retrovirus containing the transgene whereby the germcells of the embryo have the transgene chromosomally integrated therein.When the animals to be made transgenic are avian, because avianfertilized ova generally go through cell division for the first twentyhours in the oviduct, microinjection into the pronucleus of thefertilized egg is problematic due to the inaccessibility of thepronucleus. Therefore, of the methods to make transgenic animalsdescribed generally above, retrovirus infection is preferred for avianspecies, for example as described in U.S. Pat. No. 5,162,215. Ifmicro-injection is to be used with avian species, however, a publishedprocedure by Love et al., (Biotechnology, 12, Jan. 1994) can be utilizedwhereby the embryo is obtained from a sacrificed hen approximately twoand one-half hours after the laying of the previous laid egg, thetransgene is microinjected into the cytoplasm of the germinal disc andthe embryo is cultured in a host shell until maturity. When the animalsto be made transgenic are bovine or porcine, microinjection can behampered by the opacity of the ova thereby making the nuclei difficultto identify by traditional differential interference-contrastmicroscopy. To overcome this problem, the ova can first be centrifugedto segregate the pronuclei for better visualization.

The “non-human animals” of the invention include bovine, porcine, ovineand avian animals (e.g., cow, pig, sheep, chicken). The “transgenicnon-human animals” of the invention are produced by introducing“transgenes” into the germline of the non-human animal. Embryonal targetcells at various developmental stages can be used to introducetransgenes. Different methods are used depending on the stage ofdevelopment of the embryonal target cell. The zygote is the best targetfor micro-injection. The use of zygotes as is target for gene transferhas a major advantage in that in most cases the injected DNA will beincorporated into the host gene before the first cleavage (Brinster etal., Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985). As a consequence,all cells of the transgenic non-human animal will carry the incorporatedtransgene. This will in general also be reflected in the efficienttransmission of the transgene to offspring of the founder since 50% ofthe germ cells will harbor the transgene.

The term “transgenic” is used to describe an animal which includesexogenous genetic material within all of its cells. A “transgenic”animal can be produced by cross-breeding two chimeric animals whichinclude exogenous genetic material within cells used in reproduction.Twenty-five percent of the resulting offspring will be transgenic i.e.,animals which include the exogenous genetic material within all of theircells in both alleles, 50% of the resulting animals will include theexogenous genetic material within one allele and 25% will include noexogenous genetic material.

In the microinjection method useful in the practice of the subjectinvention, the transgene is digested and purified free from any vectorDNA, e.g., by gel electrophoresis. It is preferred that the transgeneinclude an operatively associated promoter which interacts with cellularproteins involved in transcription, ultimately resulting in constitutiveexpression. Promoters useful in this regard include those fromcytomegalovirus (CMV), Moloney leukemia virus (MLV), and herpes virus,as well as those from the genes encoding metallothionin, skeletal actin,P-enolpyruvate carboxylase (PEPCK), phosphoglycerate (PGK), DHFR, andthymidine kinase. Promoters for viral long terminal repeats (LTRs) suchas Rous Sarcoma Virus can also be employed. When the animals to be madetransgenic are avian, preferred promoters include those for the chickenβ-globin gene, chicken lysozyme gene, and avian leukosis virus.Constructs useful in plasmid transfection of embryonic stem cells willemploy additional regulatory elements well known in the art such asenhancer elements to stimulate transcription, splice acceptors,termination and polyadenylation signals, and ribosome binding sites topermit translation.

Retroviral infection can also be used to introduce transgene into anon-human animal, as described above. The developing non-human embryocan be cultured in vitro to the blastocyst stage. During this time, theblastomeres can be targets for retroviral infection (Jaenich, R., Proc.Natl. Acad. Sci USA 73:1260-1264, 1976). Efficient infection of theblastomeres is obtained by enzymatic treatment to remove the zonapellucida (Hogan, et al. (1986) in Manipulating the Mouse Embryo, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The viralvector system used to introduce the transgene is typically areplication-defective retro virus carrying the transgene (Jahner, etal., Proc. Natl. Acad. Sci. USA 82: 6927-6931, 1985; Van der Putten, etal., Proc. Natl. Acad. Sci USA 82: 6148-6152, 1985). Transfection iseasily and efficiently obtained by culturing the blastomeres on amonolayer of virus-producing cells (Van der Putten, supra; Stewart, etal., EMBO J. 6: 383-388, 1987). Alternatively, infection can beperformed at a later stage. Virus or virus-producing cells can beinjected into the blastocoele (D. Jahner et al., Nature 298: 623-628,1982). Most of the founders will be mosaic for the transgene sinceincorporation occurs only in a subset of the cells which formed thetransgenic nonhuman animal. Further, the founder may contain variousretro viral insertions of the transgene at different positions in thegenome which generally will segregate in the offspring. In addition, itis also possible to introduce transgenes into the germ line, albeit withlow efficiency, by intrauterine retroviral infection of the midgestationembryo (D. Jahner et al., supra).

A third type of target cell for transgene introduction is the embryonalstem cell (ES). ES cells are obtained from pre-implantation embryoscultured in vitro and fused with embryos (M. J. Evans et al., Nature292:154-156, 1981; M. O. Bradley et al., Nature 309:255-258, 1984;Gossler, et al., Proc. Natl. Acad. Sci USA 83:9065-9069, 1986; andRobertson et al., Nature 322:445-448, 1986). Transgenes can beefficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells can thereafter becombined with blastocysts from a nonhuman animal. The ES cellsthereafter colonize the embryo and contribute to the germ line of theresulting chimeric animal. (For review see Jaenisch, R., Science240:1468-1474, 1988).

“Transformed” means a cell into which (or into an ancestor of which) hasbeen introduced, by means of recombinant nucleic acid techniques, aheterologous nucleic acid molecule. “Heterologous” refers to a nucleicacid sequence that either originates from another species or is modifiedfrom either its original form or the form primarily expressed in thecell.

“Transgene” means any piece of DNA which is inserted by artifice into acell, and becomes part of the genome of the organism (i.e., eitherstably integrated or as a stable extrachromosomal element) whichdevelops from that cell. Such a transgene may include a gene which ispartly or entirely heterologous (i.e., foreign) to the transgenicorganism, or may represent a gene homologous to an endogenous gene ofthe organism. Included within this definition is a transgene created bythe providing of an RNA sequence which is transcribed into DNA and thenincorporated into the genome. The transgenes of the invention includeDNA sequences which encode phytases or polypeptides having phytaseactivity, and include polynucleotides, which may be expressed in atransgenic non-human animal. The term “transgenic” as used hereinadditionally includes any organism whose genome has been altered by invitro manipulation of the early embryo or fertilized egg or by anytransgenic technology to induce a specific gene knockout. The term “geneknockout” as used herein, refers to the targeted disruption of a gene invivo with complete loss of function that has been achieved by anytransgenic technology familiar to those in the art. In one embodiment,transgenic animals having gene knockouts are those in which the targetgene has been rendered nonfunctional by an insertion targeted to thegene to be rendered non-functional by homologous recombination. As usedherein, the term “transgenic” includes any transgenic technologyfamiliar to those in the art which can produce an organism carrying anintroduced transgene or one in which an endogenous gene has beenrendered non-functional or “knocked out.”

The transgene to be used in the practice of the subject invention is aDNA sequence comprising a sequence coding for a phytase or a polypeptidehaving phytase activity. In a one embodiment, a polynucleotide having asequence as set forth in SEQ ID NO:1 or a sequence encoding apolypeptide having a sequence as set forth in SEQ ID NO:2 is thetransgene as the term is defined herein. Where appropriate, DNAsequences that encode proteins having phytase activity but differ innucleic acid sequence due to the degeneracy of the genetic code may alsobe used herein, as may truncated forms, allelic variants andinterspecies homologues.

After an embryo has been microinjected, colonized with transfectedembryonic stem cells or infected with a retrovirus containing thetransgene (except for practice of the subject invention in avian specieswhich is addressed elsewhere herein) the embryo is implanted into theoviduct of a pseudopregnant female. The consequent progeny are testedfor incorporation of the transgene by Southern blot analysis of blood ortissue samples using transgene specific probes. PCR is particularlyuseful in this regard. Positive progeny (GO) are crossbred to produceoffspring (GI) which are analyzed for transgene expression by Northernblot analysis of tissue samples.

In one aspect, the present invention provides compositions and methodsfor increasing the phosphorous uptake in an animal (inlcuding a human, acommercially used animal, or, a transgenic animal) by decreasing theamount of phytate pollutant in the manure of the animal (e.g.,transgenic organism) by about 15%, 20%, 30%, about 20% to about 50%,40%, 50%, 50%, 50%, 50%, or more.

Animals used in the practice of the subject invention include, but arenot limited to, those animals generally regarded as domesticated animalsincluding pets (e.g., canines, felines, avian species etc.) and thoseuseful for the processing of food stuffs, i.e., avian such as meat bredand egg laying chicken and turkey, ovine such as lamb, bovine such asbeef cattle and milk cows, piscine and porcine.

In one aspect, these animals are referred to as “transgenic” when suchanimal has had a heterologous DNA sequence, or one or more additionalDNA sequences normally endogenous to the animal (collectively referredto herein as “transgenes”) chromosomally integrated into the germ cellsof the animal. The transgenic animal (including its progeny) will alsohave the transgene fortuitously integrated into the chromosomes ofsomatic cells.

In some instances it may be advantageous to deliver and express aphytase sequence of the invention locally (e.g., within a particulartissue or cell type). For example, local expression of a phytase ordigestive enzyme in the gut of an animal will assist in the digestionand uptake of, for example, phytate and phosporous, respectively. Thenucleic sequence may be directly delivered to the salivary glands,tissue and cells and/or to the epithelial cells lining the gut, forexample. Such delivery methods are known in the art and includeelectroporation, viral vectors and direct DNA uptake. Any polypeptidehaving phytase activity can be utilized in the methods of the invention(e.g., those specficially described herein, as well as those describedin other sections of the invention).

For example, nucleic acid constructs of the present invention willcomprise nucleic acid molecules in a form suitable for uptake intotarget cells within a host tissue. The nucleic acids may be in the formof bare DNA or RNA molecules, where the molecules may comprise one ormore structural genes, one or more regulatory genes, antisense strands,strands capable of triplex formation, or the like. Commonly, the nucleicacid construct will include at least one structural gene under thetranscriptional and translational control of a suitable regulatoryregion. More usually, nucleic acid constructs of the present inventionwill comprise nucleic acids incorporated in a delivery vehicle toimprove transfection efficiency, wherein the delivery vehicle will bedispersed within larger particles comprising a dried hydrophilicexcipient material.

One such delivery vehicles comprises viral vectors, such asretroviruses, adenoviruses, and adeno-associated viruses, which havebeen inactivated to prevent self-replication but which maintain thenative viral ability to bind a target host cell, deliver geneticmaterial into the cytoplasm of the target host cell, and promoteexpression of structural or other genes which have been incorporated inthe particle. Suitable retrovirus vectors for mediated gene transfer aredescribed in Kahn et al. (1992) CIRC. RES. 71:1508-1517, the disclosureof which is incorporated herein by reference. A suitable adenovirus genedelivery is described in Rosenfeld et al. (1991) SCIENCE 252:431-434,the disclosure of which is incorporated herein by reference. Bothretroviral and adenovirus delivery systems are described in Friedman(1989) SCIENCE 244:1275-1281, the disclosure of which is alsoincorporated herein by reference.

A second type of nucleic acid delivery vehicle comprises liposomaltransfection vesicles, including both anionic and cationic liposomalconstructs. The use of anionic liposomes requires that the nucleic acidsbe entrapped within the liposome. Cationic liposomes do not requirenucleic acid entrapment and instead may be formed by simple mixing ofthe nucleic acids and liposomes. The cationic liposomes avidly bind tothe negatively charged nucleic acid molecules, including both DNA andRNA, to yield complexes which give reasonable transfection efficiency inmany cell types. See, Farhood et al. (1992) BIOCHEM. BIOPHYS. ACTA.1111:239-246, the disclosure of which is incorporated herein byreference. A particularly preferred material for forming liposomalvesicles is lipofectin which is composed of an equimolar mixture ofdioleylphosphatidyl ethanolamine (DOPE) anddioleyloxypropyl-triethylammonium (DOTMA), as described in Felgner andRingold (1989) NATURE 337:387-388.

In one aspect, the invention combines these two types of deliverysystems. For example, Kahn et al. (1992), supra., teaches that aretrovirus vector may be combined in a cationic DEAE-dextran vesicle tofurther enhance transformation efficiency. It is also possible toincorporate nuclear proteins into viral and/or liposomal deliveryvesicles to even further improve transfection efficiencies. See, Kanedaet al. (1989) SCIENCE 243:375-378.

In another embodiment, the invention provides digestive aids containinga phytase, e.g., an enzyme of the invention, either as the sole activeingredient or in combination with one or more other agents and/orenzymes is provided (as described in co-pending application U.S. Ser.No. 09/580,937, entitled “Dietary Aids and Methods of Use Thereof,”filed May 25, 2000). The use of enzymes and other agents in digestiveaids of livestock or domesticated animals not only improves the animal'shealth and life expectancy but also assists in increasing the health oflivestock and in the production of foodstuffs from livestock.

Currently, some types of feed for livestock (e.g., certain poultry feed)are highly supplemented with numerous minerals (e.g., inorganicphosphorous), enzymes, growth factors, drugs, and other agents fordelivery to the livestock. These supplements replace many of thecalories and natural nutrients present in grain, for example. Byreducing or eliminating the inorganic phosphorous supplement and othersupplements (e.g., trace mineral salts, growth factors, enzymes,antibiotics) from the feed itself, the feed would be able to carry morenutrient and energy. Accordingly, the remaining diet would contain moreusable energy. For example, grain-oilseed meal diets generally containabout 3,200 kcal metabolizable energy per kilogram of diet, and mineralsalts supply no metabolizable energy. Removal of the unneeded mineralsand substitution with grain would therefore increase the usable energyin the diet. Thus, the invention can be differentiated over commonlyused phytase containing feed. For example, in one embodiment, abiocompatible material is used that is resistant to digestion by thegastrointestinal tract of an organism.

In many organisms, including, for example, poultry or birds such as, forexample, chickens, turkeys, geese, ducks, parrots, peacocks, ostriches,pheasants, quail, pigeons, emu, kiwi, loons, cockatiel, cockatoo,canaries, penguins, flamingoes, and dove, the digestive tract includes agizzard which stores and uses hard biocompatible objects (e.g., rocksand shells from shell fish) to help in the digestion of seeds or otherfeed consumed by a bird. A typical digestive tract of this generalfamily of organisms, includes the esophagus which contains a pouch,called a crop, where food is stored for a brief period of time. From thecrop, food moves down into the true stomach, or proventriculus, wherehydrochloric acid and pepsin starts the process of digestion. Next, foodmoves into the gizzard, which is oval shaped and thick walled withpowerful muscles. The chief function of the gizzard is to grind or crushfood particles—a process which is aided by the bird swallowing smallamounts of fine gravel or grit. From the gizzard, food moves into theduodenum. The small intestine of birds is similar to mammals. There aretwo blind pouches or ceca, about 4-6 inches in length at the junction ofthe small and large intestine. The large intestine is short, consistingmostly of the rectum about 3-4 inches in length. The rectum empties intothe cloaca and feces are excreted through the vent.

Hard, biocompatible objects consumed (or otherwise introduced) andpresent in the gizzard provide a useful vector for delivery of variousenzymatic, chemical, therapeutic and antibiotic agents. These hardsubstances have a life span of a few hours to a few days and are passedafter a period of time. Accordingly, the invention provides coated,impregnated (e.g., impregnated matrix and membranes) modified dietaryaids for delivery of useful digestive or therapeutic agents to anorganism. Such dietary aids include objects which are typically ingestedby an organism to assist in digestion within the gizzard (e.g., rocks orgrit). The invention provides biocompatible objects that have coatedthereon or impregnated therein agents useful as a digestive aid for anorganism or for the delivery of a therapeutic or medicinal agent orchemical.

In a first embodiment, the invention provides a dietary aid, having abiocompatible composition designed for release of an agent that assistsin digestion, wherein the biocompatible composition is designed for oralconsumption and release in the digestive tract (e.g., the gizzard) of anorganism. “Biocompatible” means that the substance, upon contact with ahost organism (e.g., a bird), does not elicit a detrimental responsesufficient to result in the rejection of the substance or to render thesubstance inoperable. Such inoperability may occur, for example, byformation of a fibrotic structure around the substance limitingdiffusion of impregnated agents to the host organism therein or asubstance which results in an increase in mortality or morbidity in theorganism due to toxicity or infection. A biocompatible substance may benon-biodegradable or biodegradable. In one embodiment, the biocompatiblecomposition is resistant to degradation or digestion by thegastrointestinal tract. In another embodiment, the biocompatiblecomposition has the consistency of a rock or stone.

A non-biodegradable material useful in the invention is one that allowsattachment or impregnation of a dietary agent. Such non-biodegradablematerials include, for example, thermoplastics, such as acrylic,modacrylic, polyamide, polycarbonate, polyester, polyethylene,polypropylene, polystyrene, polysulfone, polyethersulfone, andpolyvinylidene fluoride. Elastomers are also useful materials andinclude, for example, polyamide, polyester, polyethylene, polypropylene,polystyrene, polyurethane, polyvinyl alcohol and silicone (e.g.,silicone based or containing silica). The invention provides that thebiocompatible composition can contain a plurality of such materials,which can be, e.g., admixed or layered to form blends, copolymers orcombinations thereof.

As used herein, a “biodegradable” material means that the compositionwill erode or degrade in vivo to form smaller chemical species.Degradation may occur, for example, by enzymatic, chemical or physicalprocesses. Suitable biodegradable materials contemplated for use in theinvention include poly(lactide)s, poly(glycolide)s, poly(lactic acid)s,poly(glycolic acid)s, polyanhydrides, polyorthoesters, polyetheresters,polycaprolactone, polyesteramides, polycarbonate, polycyanoacrylate,polyurethanes, polyacrylate. Such materials can be admixed or layered toform blends, copolymers or combinations thereof.

It is contemplated that a number of different biocompatible substancesmay be ingested or otherwise provided to the same organismsimultaneously, or in various combinations (e.g., one material beforethe other). In addition, the biocompatible substance may be designed forslow passage through the digestive tract. For example, large or fattysubstances tend to move more slowly through the digestive tract,accordingly, a biocompatible material having a large size to preventrapid passing in the digestive tract can be used. Such large substancescan be a combination of non-biodegradable and biodegradable substances.For example, a small non-biodegradable substance can be encompassed by abiodegradable substance such that over a period of time thebiodegradable portion will be degraded allowing the non-biodegradableportion to pass through the digestive trace. In addition, it isrecognized that any number of flavorings can be provided to thebiocompatible substance to assist in consumption.

Any number of agents alone or in combination with other agents can becoated on the biocompatible substance including polypeptides (e.g.,enzymes, antibodies, cytokines or therapeutic small molecules), andantibiotics, for example. Examples of particular useful agents arelisted in Table 1 and 2, below. It is also contemplated that cells canbe encapsulated into the biocompatible material of the invention andused to deliver the enzymes or therapeutics. For example, poroussubstances can be designed that have pores large enough for cells togrow in and through and that these porous materials can then be takeninto the digestive tract. For example, the biocompatible substance canbe comprised of a plurality of microfloral environments (e.g., differentporosity, pH etc.) that provide support for a plurality of cell types.The cells can be genetically engineered to deliver a particular drug,enzyme or chemical to the organism. The cells can be eukaryotic orprokaryotic. TABLE 1 Treatment Class Chemical Description AntibioticsAmoxycillin and Its Treatment Against Bacterial Combination DiseasesCaused By Gram + Mastox Injection and Gram − Bacteria (Amoxycillin andCloxacillin) Ampicillin and Its Combination Treatment Against BacterialBiolox Injection Diseases Caused By Gram + (Ampicillin and Cloxacillin)And Gram − Bacteria. Nitrofurazone + Urea Treatment Of GenitalInfections Nefrea Bolus Trimethoprim + Treatment Of Respiratory TractSulphamethoxazole Infections, Gastro Intestinal Trizol Bolus TractInfections, Urino- Genital Infections. Metronidazole and FurazolidoneTreatment Of Bacterial And Metofur Bolus Protozoal Diseases.Phthalylsulphathiazole, Pectin Treatment Of Bacterial And and KaolinNon-Specific Diarrhoea, Pectolin Bacillary Dysentry And Calf BolusScours. Suspension Antihelmintics Ectoparasiticide Ectoparasiticide andAntiseptic Germex Ointment (Gamma Benzene Hexachloride, ProflavinHemisulphate and Cetrimide) Endoparasiticides > Albendazole PreventionAnd Treatment Of and Its Combination Roundworm, Tapeworm and Alben(Albendazole) Fluke Infestations Suspension (Albendazole 2.5%) PlusSuspension (Albendazole 5%) Forte Bolus (Albendazole 1.5 Gm.) Tablet(Albendazole 600 Mg.) Powder(Albendazole 5%, 15%) Alpraz (Albendazoleand Prevention And Treatment Of Praziquantel)Tablet Roundworm andTapeworm Infestation In Canines and Felines. Oxyclozanide and ItsPrevention and Treatment Of Combination Fluke Infestations Clozan(Oxyclozanide) Bolus, Suspension Tetzan (Oxyclozanide and Prevention andTreatment Of Tetramisole Hcl) Bolus, Roundworm and Fluke SuspensionInfestations Fluzan (Oxyclozanide and Prevention and Treatment OfLevamisole Hcl) Bolus, Roundworm Infestations and Suspension IncreasingImmunity Levamisole Prevention and Treatment Of Nemasol InjectionRoundworm Infestations and Wormnil Powder Increasing Immunity.Fenbendazole Prevention And Treatment of Fenzole Roundworm and TapewormTablet (Fenbendazole 150 Mg.) Infestations Bolus (Fenbendazole 1.5 Gm.)Powder (Fenbendazole 2.5% W/W) Tonics Vitamin B Complex, Amino TreatmentOf Anorexia, Acids and Liver Extract Hepatitis, Debility, NeuralgicHeptogen Injection Convulsions Emaciation and Stunted Growth. CalciumLevulinate With Vit. B₁₂ Prevention and treatment of and Vit D₃hypocalcaemia, supportive Hylactin Injection therapy in sick conditions(especially hypothermia) and treatment of early stages of rickets.Animal Feed Supplements Essential Minerals, Selenium and Treatment OfAnoestrus Causing Vitamin E Infertility and Repeat Breeding GynolactinBolus In Dairy Animals and Horses. Essential Minerals, Vitamin E,Infertility, Improper Lactation, and Iodine Decreased Immunity, StuntedHylactin Powder Growth and Debility. Essential Electrolytes WithDiarrhoea, Dehydration, Prior to Vitamin C and after Transportation, InElectra - C Powder Extreme temperatures (High Or Low) and otherConditions of stress. Pyrenox Plus (Diclofenac Treatment Of Mastitis,Pyrexia Sodium + Paracetamol) Bolus, Post Surgical Pain and Injection.Inflammation, Prolapse Of Uterus, Lameness and Arthritis.

TABLE 2 Therapeutic Formulations Product Description Acutrim ®Once-daily appetite suppressant tablets. (phenylpropanolamine) TheBaxter ® Infusor For controlled intravenous delivery of anticoagulants,antibiotics, chemotherapeutic agents, and other widely used drugs.Catapres-TTS ® (clonidine Once-weekly transdermal system for thetreatment of transdermal therapeutic system) hypertension. Covera HS3(verapamil Once-daily Controlled-Onset Extended-Release (COER-24)hydrochloride) tablets for the treatment of hypertension and anginapectoris. DynaCirc CR ® (isradipine) Once-daily extended release tabletsfor the treatment of hypertension. Efidac 24 ® (chlorpheniramineOnce-daily extended release tablets for the relief of allergy maleate)symptoms. Estraderm ® Twice-weekly transdermal system for treatingcertain (estradiol transdermal system) postmenopausal symptoms andpreventing osteoporosis Glucotrol XL ® (glipizide) Once-daily extendedrelease tablets used as an adjunct to diet for the control ofhyperglycemia in patients with non-insulin- dependent diabetes mellitus.IVOMEC SR ® Bolus Ruminal delivery system for season-long control ofmajor (ivermectin) internal and external parasites in cattle. MinipressXL ® (prazosin) Once-daily extended release tablets for the treatment ofhypertension. NicoDerm ® CQ ™ (nicotine Transdermal system used as aonce-daily aid to smoking transdermal system) cessation for relief ofnicotine withdrawal symptoms. Procardia XL ® (nifedipine) Once-dailyextended release tablets for the treatment of angina and hypertension.Sudafed ® 24 Hour Once-daily nasal decongestant for relief of colds,sinusitis, (pseudoephedrine) hay fever and other respiratory allergies.Transderm-Nitro ® (nitroglycerin Once-daily transdermal system for theprevention of angina transdermal system) pectoris due to coronary arterydisease. Transderm Scop ® (scopolamin Transdermal system for theprevention of nausea and transdermal system) vomiting associated withmotion sickness. Volmax (albuterol) Extended release tablets for reliefof bronchospasm in patients with reversible obstructive airway disease.Actisite ® (tetracycline hydrochloride) Periodontal fiber used as anadjunct to scaling and root planing for reduction of pocket depth andbleeding on probing in patients with adult periodontitis. ALZET ®Osmotic pumps for laboratory research. Amphotec ® (amphotericin BAMPHOTEC ® is a fungicidal treatment for invasive cholesteryl sulfatecomplex for aspergillosis in patients where renal impairment orinjection) unacceptable toxicity precludes use of amphotericin B ineffective doses and in patients with invasive aspergillosis where prioramphotericin B therapy has failed. BiCitra ® (sodium citrate andAlkalinizing agent used in those conditions where long-term citric acid)maintenance of alkaline urine is desirable. Ditropan ® (oxybutyninchloride) For the relief of symptoms of bladder instability associatedwith uninhibited neurogenic or reflex neurogenic bladder (i.e., urgency,frequency, urinary leakage, urge incontinence, dysuria). Ditropan ® XL(oxybutynin is a once-daily controlled-release tablet indicated for thechloride) treatment of overactive bladder with symptoms of urge urinaryincontinence, urgency and frequency. DOXIL ® (doxorubicin HCl liposomeinjection) Duragesic ® (fentanyl transdermal 72-hour transdermal systemfor management of chronic pain system) CII in patients who requirecontinuous opioid analgesia for pain that cannot be managed by lessermeans such as acetaminophen-opioid combinations, non-steroidalanalgesics, or PRN dosing with short-acting opioids. Elmiron ® (pentosanpolysulfate Indicated for the relief of bladder pain or discomfortsodium) associated with interstitial cystitis. ENACT AirWatch ™ Anasthma monitoring and management system. Ethyol ® (amifostine) Indicatedto reduce the cumulative renal toxicity associated with repeatedadministration of cisplatin in patients with advanced ovarian cancer ornon-small cell lung cancer. Indicated to reduce the incidence ofmoderate to severe xerostomia in patients undergoing post-operativeradiation treatment for head and neck cancer, where the radiation portincludes a substantial portion of the parotid glands. Mycelex ® Troche(clotrimazole) For the local treatment of oropharyngeal candidiasis.Also indicated prophylactically to reduce the incidence of oropharyngealcandidiasis in patients immunocompromised by conditions that includechemotherapy, radiotherapy, or steroid therapy utilized in the treatmentof leukemia, solid tumors, or renal transplantation. Neutra-Phos ®(potassium and a dietary/nutritional supplement sodium phosphate)PolyCitra ® -K Oral Solution Alkalinizing agent useful in thoseconditions where long- and PolyCitra ® -K Crystals term maintenance ofan alkaline urine is desirable, such as in (potassium citrate and citricacid) patents with uric acid and cystine calculi of the urinary tract,especially when the administration of sodium salts is undesirable orcontraindicated PolyCitra ® -K Syrup and LC Alkalinizing agent useful inthose conditions where long- (tricitrates) term maintenance of analkaline urine is desirable, such as in patients with uric acid andcystine calculi of the urinary tract. Progestasert ® (progesterone)Intrauterine Progesterone Contraceptive System Testoderm ® Testoderm ®with Testosterone Transdermal System Adhesive and Testoderm ® TTS TheTestoderm ® products are indicated for replacement CIII therapy in malesfor conditions associated with a deficiency or absence of endogenoustestosterone: (1) Primary hypogonadism (congenital or acquired) or (2)Hypogonadotropic hypogonadism (congenital or acquired). Viadur ™(leuprolide acetate Once-yearly implant for the palliative treatment ofprostate implant) cancer

Certain agents can be designed to become active or in activated undercertain conditions (e.g., at certain pH's, in the presence of anactivating agent etc.). In addition, it may be advantageous to usepro-enzymes in the compositions of the invention. For example, apro-enzymes can be activated by a protease (e.g., a salivary proteasethat is present in the digestive tract or is artificially introducedinto the digestive tract of an organism). It is contemplated that theagents delivered by the biocompatible compositions of the invention canbe activated or inactivated by the addition of an activating agent whichmay be ingested by, or otherwise delivered to, the organism. Anothermechanism for control of the agent in the digestive tract is anenvironment sensitive agent that is activated in the proper digestivecompartment. For example, an agent may be inactive at low pH but activeat neutral pH. Accordingly, the agent would be inactive in the gut butactive in the intestinal tract. Alternatively, the agent can becomeactive in response to the presence of a microorganism specific factor(e.g., microorganisms present in the intestine).

In summary, the potential benefits of the present invention include, forexample, (1) reduction in or possible elimination of the need formineral supplements (e.g., inorganic phosphorous supplements), enzymes,or therapeutic drugs for animal (including fish) from the daily feed orgrain thereby increasing the amount of calories and nutrients present inthe feed, and (2) increased health and growth of domestic andnon-domestic animals including, for example, poultry, porcine, bovine,equine, canine, and feline animals.

A large number of enzymes can be used in the methods and compositions ofthe present invention. These enzymes include enzymes necessary forproper digestion of consumed foods, or for proper metabolism, activationor derivation of chemicals, prodrugs or other agents or compoundsdelivered to the animal via the digestive tract. Examples of enzymesthat can be delivered or incorporated into the compositions of theinvention, include, for example, feed enhancing enzymes selected fromthe group consisting of α-galactosidases, β-galactosidases, inparticular lactases, phytases, β-glucanases, in particularendo-β-1,4-glucanases and endo-β-1,3(4)-glucanases, cellulases,xylosidases, galactanases, in particular arabinogalactanendo-1,4-β-galactosidases and arabinogalactan endo-1,3-β-galactosidases,endoglucanases, in particular endo-1,2-β-glucanase,endo-1,3-α-glucanase, and endo-1,3-β-glucanase, pectin degradingenzymes, in particular pectinases, pectinesterases, pectin lyases,polygalacturonases, arabinanases, rhamnogalacturonases,rhamnogalacturonan acetyl esterases, rhamnogalacturonan-α-rhamnosidase,pectate lyases, and α-galacturonisidases, mannanases, β-mannosidases,mannan acetyl esterases, xylan acetyl esterases, proteases, xylanases,arabinoxylanases and lipolytic enzymes such as lipases, phospholipasesand cutinases. Phytases as set forth in SEQ ID NO:1 and 2 and in Table 3below are preferred. The sequences described in Table 3 are SEQ ID NO:1and 2 having the amino acid substitutions and nucleotide substitutionsas described therein. TABLE 3 Nuc. Designation Source AA seq Sequence E.coli B E. coli B S10; P26; (reference) D176; M298; A299; G312; I428868PH1 Bison E. coli I428T 872PH1 Kangaroo rat D176G; G312S GAC(176)GGC;E. coli M298K; A299T GGT(312)AGT; ATG(298)AAG; GCA(299)ACA 875PH2 E.coli W A160S; D176G; GCG(160)TCG; M298K; A299T GAC(176)GGC; ATG(298)AAG;GCA(299)ACA 873PH1 Calf E. coli 1428R E. coli B E. coli B K298M; T299AAAG(298)ATG; ACA(299)GCA K12 appA E. coli K12 M298K; A299T ATG(298)AAG;GCA(299)ACA

The enzymes used in the invention (including the phytases of theinvention) can be modified to enhance their activity, delivery,activation and degradation. Such modifications can be performed in vivoor in vitro and use methods and processes generally known in the art asdescribed more fully below. Such methodology generally usespolynucleotide or polypeptide sequences that are either synthesized byautomated machines or are cloned, expressed, or manipulated byrecombinant DNA techniques.

In a preferred embodiment, the enzyme used in the compositions (e.g., adietary aid) of the present invention is a phytase enzyme (e.g., aphytase of the invention) which is stable to heat and is heat resistantand catalyzes the enzymatic hydrolysis of phytate, i.e., the enzyme isable to renature and regain activity after a brief (i.e., 5 to 30seconds), or longer period, for example, minutes or hours, exposure totemperatures of above 50° C.

A “feed” and a “food,” respectively, means any natural or artificialdiet, meal or the like or components of such meals intended or suitablefor being eaten, taken in, digested, by an animal and a human being,respectively. “Dietary Aid,” as used herein, denotes, for example, acomposition containing agents that provide a therapeutic or digestiveagent to an animal or organism. A “dietary aid,” typically is not asource of caloric intake for an organism, in other words, a dietary aidtypically is not a source of energy for the organism, but rather is acomposition which is taken in addition to typical “feed” or “food”.

An agent or enzyme (e.g., a phytase) may exert its effect in vitro or invivo, i.e. before intake or in the stomach or gizzard of the organism,respectively. Also a combined action is possible.

Although any enzyme may be incorporated into a dietary aid of theinvention, reference is made herein to phytase as an exemplification ofthe methods and compositions of the invention. A dietary aid of theinvention includes an enzyme (e.g., a phytase of the invention, e.g., apolypeptide having a sequence as set forth in SEQ ID NO:10). In oneaspect, a dietary aid of the invention containing a phytase compositionis liquid or dry.

Liquid compositions need not contain anything more than the enzyme (e.g.a phytase), preferably in a highly purified form. Usually, however, astabilizer such as glycerol, sorbitol or mono propylen glycol is alsoadded. The liquid composition may also comprise other additives, such assalts, sugars, preservatives, pH-adjusting agents, proteins, phytate (aphytase substrate). Typical liquid compositions are aqueous or oil-basedslurries. The liquid compositions can be added to a biocompatiblecomposition for slow release. Preferably the enzyme is added to adietary aid composition that is a biocompatible material (e.g.,biodegradable or non-biodegradable) and includes the addition ofrecombinant cells into, for example, porous microbeads.

Dry compositions may be spray dried compositions, in which case thecomposition need not contain anything more than the enzyme in a dryform. Usually, however, dry compositions are so-called granulates whichmay readily be mixed with a food or feed components, or more preferably,form a component of a pre-mix. The particle size of the enzymegranulates preferably is compatible with that of the other components ofthe mixture. This provides a safe and convenient means of incorporatingenzymes into animal feed. Preferably the granulates are biocompatibleand more preferably the biocompatible granulates are non-biodegradable.

Agglomeration granulates coated by an enzyme can be prepared usingagglomeration techniques in a high shear mixer Absorption granulates areprepared by having cores of a carrier material to absorb be coated bythe enzyme. Preferably the carrier material is a biocompatiblenon-biodegradable material that simulates the role of stones or grit inthe gizzard of an animal. Typical filler materials used in agglomerationtechniques include salts, such as disodium sulphate. Other fillers arekaolin, talc, magnesium aluminum silicate and cellulose fibres.Optionally, binders such as dextrins are also included in agglomerationgranulates. The carrier materials can be any biocompatible materialincluding biodegradable and non-biodegradable materials (e.g., rocks,stones, ceramics, various polymers). Optionally, the granulates arecoated with a coating mixture. Such mixture comprises coating agents,preferably hydrophobic coating agents, such as hydrogenated palm oil andbeef tallow, and if desired other additives, such as calcium carbonateor kaolin.

Additionally, the dietary aid compositions of the invention (e.g.,phytase dietary aid compositions of the invention) may contain othersubstituents such as colouring agents, aroma compounds, stabilizers,vitamins, minerals, other feed or food enhancing enzymes etc. A typicaladditive usually comprises one or more compounds such as vitamins,minerals or feed enhancing enzymes and suitable carriers and/orexcipients.

In one embodiment, the dietary aid compositions of the inventionadditionally comprises an effective amount of one or more feed enhancingenzymes, in particular feed enhancing enzymes selected from the groupconsisting of α-galactosidases, β-galactosidases, in particularlactases, other phytases, β-glucanases, in particularendo-β-1,4-glucanases and endo-β-1,3(4)-glucanases, cellulases,xylosidases, galactanases, in particular arabinogalactanendo-1,4-β-galactosidases and arabinogalactan endo-1,3-β-galactosidases,endoglucanases, in particular endo-1,2-β-glucanase,endo-1,3-α-glucanase, and endo-1,3-β-glucanase, pectin degradingenzymes, in particular pectinases, pectinesterases, pectin lyases,polygalacturonases, arabinanases, rhamnogalacturonases,rhamnogalacturonan acetyl esterases, rhamnogalacturonan-α-rhamnosidase,pectate lyases, and α-galacturonisidases, mannanases, β-mannosidases,mannan acetyl esterases, xylan acetyl esterases, proteases, xylanases,arabinoxylanases and lipolytic enzymes such as lipases, phospholipasesand cutinases.

The animal dietary aid of the invention is supplemented to themono-gastric animal before or simultaneously with the diet. In oneembodiment, the dietary aid of the invention is supplemented to themono-gastric animal simultaneously with the diet. In another embodiment,the dietary aid is added to the diet in the form of a granulate or astabilized liquid.

Phytase activity is expressed as FYT, FTU, PU and U. All are same andone unit of phytase is defined as the amount of enzyme that liberates 1micromole of inorganic phosphorus per minute from 1.5 millimole sodiumphytate solution at 37° C. and pH 5.5. An effective amount of an enzymein a dietary aid of the invention is from about 10-20,000; preferablyfrom about 10 to 15,000, more preferably from about 10 to 10,000, inparticular from about 100 to 5,000, especially from about 100 to about2,000 FYT/kg dietary aid. In one aspect, phytases (e.g., a phytase ofthe invention) is administered (e.g., fed to the animal or individual)at a dosage of about 500 FYT per kg feed or 100 grams of phytase per tonfeed with an activity of 5,000 FYT/gm, or 200 grams of phytase per tonfeed with an activity of 2,500 FYT/gm. Other dosages can beadministered, depending on the animal whose diet is to supplemented, andfor what reason; for example, for poultry layers, add about 450 to 500FYT per kg feed or 180 to 200 grams of phytase with an activity of 2,500FYT/gm of phytase per ton feed.

Examples of other specific uses of a phytase of the invention is in soyprocessing and in the manufacture of inositol or derivatives thereof.

The invention also relates to a method for reducing phytate levels inanimal manure, wherein the animal is fed a dietary aid containing aneffective amount of the phytase of the invention. As stated in thebeginning of the present application one important effect thereof is toreduce the phosphate pollution in the environment.

In another embodiment, the dietary aid of the invention is a magneticcarrier. For example, a magnetic carrier containing an enzyme (e.g., aphytase of the invention) distributed in, on or through a magneticcarrier (e.g., a porous magnetic bead), can be distributed over an areahigh in phytate and collected by magnets after a period of time. Suchdistribution and recollection of beads reduces additional pollution andallows for reuse of the beads. In addition, use of such magnetic beadsin vivo allows for the localization of the dietary aid to a point in thedigestive tract where, for example, phytase activity can be carried out.For example, a dietary aid of the invention containing digestive enzymes(e.g., a phytase) can be localized to the gizzard of the animal byjuxtapositioning a magnet next to the gizzard of the animal after theanimal consumes a dietary aid of magnetic carriers. The magnet can beremoved after a period of time allowing the dietary aid to pass throughthe digestive tract. In addition, the magnetic carriers are suitable forremoval from the organism after sacrificing or to aid in collection.

When the dietary aid of the invention is a porous particle, suchparticles can be impregnated by a substance with which to form a slowrelease particle. Such slow release particles may be prepared not onlyby impregnating the porous particles with the substance it is desired torelease, but also by first dissolving the desired substance in the firstdispersion phase. In this case, slow release particles prepared by themethod in which the substance to be released is first dissolved in thefirst dispersion phase are also within the scope and spirit of theinvention. The porous hollow particles may, for example, be impregnatedby a slow release substance such as a medicine, agricultural chemical orenzyme. In particular, when porous hollow particles impregnated by anenzyme are made of a biodegradable polymers, the particles themselvesmay be used as an agricultural chemical or fertilizer, and they have noadverse effect on the environment. In one embodiment the porousparticles are magnetic in nature.

The porous hollow particles may be used as a bioreactor support, inparticular an enzyme support. Therefore, it is advantageous to preparethe dietary aid utilizing a method for slow release, for instance byencapsulating the enzyme of agent in a microvesicle, such as a liposome,from which the dose is released over the course of several days,preferably between about 3 to 20 days. Alternatively, the agent (e.g.,an enzyme) can be formulated for slow release, such as incorporationinto a slow release polymer from which the agent (e.g., enzyme) isslowly released over the course of several days, for example from 2 to30 days and can range up to the life of the animal.

The invention also provides compositions, e.g., dietary aids, includingphytases, such as the enzymes of the invention, in the form ofliposomal-formulated compositions. Liposomes of the invention can bederived from phospholipids or other lipid substances. Liposomes can beformed by mono- or multilamellar hydrated liquid crystals that aredispersed in an aqueous medium. Any non-toxic, physiologicallyacceptable and metabolizable lipid capable of forming liposomes can beused. The present compositions of the invention in liposome form cancontain stabilizers, preservatives, excipients, and the like in additionto the agent. The preferred lipids are the phospholipids and thephosphatidyl cholines (lecithins), both natural and synthetic. Methodsto form liposomes are known in the art. See, for example, Prescott, Ed.,Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y.(1976), p. 33 et seq.

Also within the scope of the invention is the use of a phytase of theinvention during the preparation of food or feed preparations oradditives, e.g., dietary additives or dietary aids. For example, in oneaspect, the phytase exerts its phytase activity during the manufactureonly and is not active in the final food or feed product. This aspect isrelevant for instance in dough making and baking. Accordingly, phytaseor recombinant yeast expressing phytase can be impregnated in, on orthrough magnetic carriers, distributed in the dough or food medium, andretrieved by magnets.

The dietary aid of the invention may be administered alone to animals ina biocompatible (e.g., a biodegradable or non-biodegradable) carrier orin combination with other digestion additive agents. The dietary aid ofthe invention thereof can be readily administered as a top dressing orby mixing them directly into animal feed or provided separate from thefeed, by separate oral dosage, by injection or by transdermal means orin combination with other growth related edible compounds, theproportions of each of the compounds in the combination being dependentupon the particular organism or problem being addressed and the degreeof response desired. It should be understood that the specific dietarydosage administered in any given case will be adjusted in accordancewith the specific compounds being administered, the problem to betreated, the condition of the subject and the other relevant facts thatmay modify the activity of the effective ingredient or the response ofthe subject, as is well known by those skilled in the art. Li general,either a single daily dose or divided daily dosages may be employed, asis well known in the art.

If administered separately from the animal feed, forms of the dietaryaid can be prepared by combining them with non-toxic pharmaceuticallyacceptable edible carriers to make either immediate release or slowrelease formulations, as is well known in the art. Such edible carriersmay be either solid or liquid such as, for example, corn starch,lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil andpropylene glycol. If a solid carrier is used the dosage form of thecompounds may be tablets, capsules, powders, troches or lozenges or topdressing as micro-dispersable forms. If a liquid carrier is used, softgelatin capsules, or syrup or liquid suspensions, emulsions or solutionsmay be the dosage form. The dosage forms may also contain adjuvants,such as preserving, stabilizing, wetting or emulsifying agents, solutionpromoters, etc. They may also contain other therapeutically valuablesubstances.

Thus, significant advantages of the invention can include forexample, 1) ease of manufacture of the active ingredient loadedbiocompatible compositions; 2) versatility as it relates to the class ofpolymers and/or active ingredients which may be utilized; 3) higheryields and loading efficiencies; and 4) the provision of sustainedrelease formulations that release active, intact active agents in vivo,thus providing for controlled release of an active agent over anextended period of time. In addition, another advantage is due to thelocal delivery of the agent with in the digestive tract (e.g., thegizzard) of the organism. As used herein the phrase “contained within”denotes a method for formulating an agent into a composition useful forcontrolled release, over an extended period of time.

In the sustained-release or slow release compositions of the invention,an effective amount of an agent (e.g., an enzyme or antibiotic) will beutilized. As used herein, sustained release or slow release refers tothe gradual release of an agent from a biocompatible material, over anextended period of time. The sustained release can be continuous ordiscontinuous, linear or non-linear, and this can be accomplished usingone or more biodegradable or non-biodegradable compositions, drugloadings, selection of excipients, or other modifications. However, itis to be recognized that it may be desirable to provide for a “fast”release composition, that provides for rapid release once consumed bythe organism. It is also to be understood that “release” does notnecessarily mean that the agent is released from the biocompatiblecarrier. Rather in one embodiment, the slow release encompasses slowactivation or continual activation of an agent present on thebiocompatible composition. For example, a phytase need not be releasedfrom the biocompatible composition to be effective. In this embodiment,the phytase is immobilized on the biocompatible composition.

The animal feed may be any protein-containing organic meal normallyemployed to meet the dietary requirements of animals. Many of suchprotein-containing meals are typically primarily composed of corn,soybean meal or a corn/soybean meal mix. For example, typicalcommercially available products fed to fowl include Egg Maker Complete,a poultry feed product of Land O'Lakes AG Services, as well as CountryGame & Turkey Grower a product of Agway, Inc. (see also The Emu Farmer'sHandbook by Phillip Minnaar and Maria Minnaar). Both of thesecommercially available products are typical examples of animal feedswith which the present dietary aid and/or the enzyme phytase may beincorporated to reduce or eliminate the amount of supplementalphosphorus, zinc, manganese and iron intake required in suchcompositions.

The present invention is applicable to the diet of numerous animals,which herein is defined as including mammals (including humans), fowland fish. In particular, the diet may be employed with commerciallysignificant mammals such as pigs, cattle, sheep, goats, laboratoryrodents (rats, mice, hamsters and gerbils), fur-bearing animals such asmink and fox, and zoo animals such as monkeys and apes, as well asdomestic mammals such as cats and dogs. Typical commercially significantavian species include chickens, turkeys, ducks, geese, pheasants, emu,ostrich, loons, kiwi, doves, parrots, cockatiel, cockatoo, canaries,penguins, flamingoes, and quail. Commercially farmed fish such as troutwould also benefit from the dietary aids disclosed herein. Other fishthat can benefit include, for example, fish (especially in an aquariumor acquaculture environment, e.g., tropical fish), goldfish and otherornamental carp, catfish, trout, salmon, shark, ray, flounder, sole,tilapia, medaka, guppy, molly, platyfish, swordtail, zebrafish, andloach.

Unless otherwise stated, transformation was performed as described inthe method of Sambrook, Fritsch and Maniatus, 1989.

Human and Animal Dietary Supplements

The invention provides novel dietary formulation, supplements andadditives for humans and animals and methods for diet supplementationcomprising phytases, e.g., a phytase of the invention (e.g., a phytasehaving a sequence identity of at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97.5%, 98%, 98.5%, 99%, 99.5%, or more, or complete (100%) sequenceidentity (i.e., homology) to SEQ ID NO:2 (a phytase polypeptide); SEQ IDNO:10 (a phytase polypeptide); a polypeptide having sequence as setforth in SEQ ID NO:8 and having at least one, or all, of the amino acidmodifications W68E, Q84W, A95P, K97C, S168E, R181Y, N226C, Y277D,wherein the polypeptide has phytase activity) or other phytases, forexample, the E. coli appA “wild type” phytase-encoding SEQ ID NO:7, or,a polypeptide sequence of SEQ ID NO:2 or the E. coli appA “wild type”phytase SEQ ID NO:8.

For example, the invention provides novel formulations and dietarysupplements and additives, and methods for diet supplementation forcertain diets, e.g., Atkins' diet, vegetarian diet, macrobiotic diet,vegan diet or regional diets, e.g., developing world diets. Foodsassociated with certain elective diets, such as Atkins, vegetarian,macrobiotic, vegan or regional diets (for example, developing worlddiets) emphasize certain food categories, such as proteins and fats,soy, etc., or they rely on indigenous crops, e.g., cereals, rice, beans,and the like as substantial or sole contributors to individualnutrition. Many of these cereal based crops have elevated (3 to 10 fold)levels of phytic acid. Processed food products such as soy proteinhydrolysate and others appear to retain elevated levels of phytic acidand their inclusion as a protein source to nutrient bars, powders andother foods or food supplements and ingredients increases the phyticacid load experienced by individuals who practice these diets.

Preventing and Reversing Bone Loss

The invention also provides novel pharmaceutical and dietaryformulations to be used as supplements and additives, and methods fordietary supplementation, comprising phytases, e.g., any phytase,including a phytase of the invention, for individuals predisposed tobone loss, individuals with bone loss, and individuals with certainmedical conditions, e.g., osteoporosis, cachexia, and medicaltreatments, such as chemotherapies, which can compromise the properuptake or utilization of essential nutrients. The methods andcompositions of the invention can be used alone or in combination withother supplements or treatment regimens, including with medications andthe like. For example, the formulations, dietary supplements and methodsfor diet supplementation can be administered with other dietarysupplements or medications for the treatment or prevention ofosteoporosis, e.g., with vitamin D3 and/or calcium (which are proven inpreventing bone loss). In one aspect, the invention provides aformulation comprising a phytase, e.g., any phytase or a phytase of theinvention, and vitamin D3 and/or calcium. In one aspect, the inventionprovides a formulation comprising a phytase, e.g., any phytase or aphytase of the invention, for preventing bone loss. In one aspect, theinvention provides a formulation comprising a phytase, e.g., any phytaseor a phytase of the invention, for reversing bone loss.

The formulation can be in the form of a pharmaceutical composition, or,can be an additive to a pharmaceutical, either of which can be inliquid, solid, powder, lotion, spray or aerosol forms. Pharmaceuticalcompositions and formulations of the invention for oral administrationcan be formulated using pharmaceutically acceptable carriers well knownin the art in appropriate and suitable dosages. Such carriers enable thepharmaceuticals to be formulated in unit dosage forms as tablets, pills,powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries,suspensions, etc., suitable for ingestion by the patient. Pharmaceuticalpreparations for oral use can be formulated as a solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable additional compounds, if desired, toobtain tablets or dragee cores. Suitable solid excipients arecarbohydrate or protein fillers include, e.g., sugars, includinglactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,potato, or other plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; andgums including arabic and tragacanth; and proteins, e.g., gelatin andcollagen. Disintegrating or solubilizing agents may be added, such asthe cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

The invention provides aqueous suspensions comprising a phytase, e.g., aphytase of the invention, in admixture with excipients suitable for themanufacture of aqueous suspensions. Such excipients include a suspendingagent, such as sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia, and dispersing or wetting agents such as anaturally occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethylene oxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensationproduct of ethylene oxide with a partial ester derived from fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate).The aqueous suspension can also contain one or more preservatives suchas ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

The dosage regimen also takes into consideration pharmacokineticsparameters well known in the art, i.e., the active agents' rate ofabsorption, bioavailability, metabolism, clearance, and the like (see,e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617;Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995)Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108;the latest edition of Remington, The Science and Practice of Pharmacy20^(th) Ed. Lippincott Williams & Wilkins). The state of the art allowsthe clinician to determine the dosage regimen for each individualpatient, active agent and disease or condition treated. Guidelinesprovided for similar compositions used as pharmaceuticals can be used asguidance to determine the dosage regiment, i.e., dose schedule anddosage levels, administered practicing the methods of the invention(e.g., reversing bone loss, or, preventing bone loss) are appropriateand correct.

Physical Training Supplements

The invention also provides novel dietary supplements and additives, andmethods of using them, comprising phytases, e.g., any phytase, or, aphytase of the invention, for individuals undergoing athletic or otherintense physical training, e.g., training for soldiers. Athletictraining and hyperexertion can deplete essential nutrients and requiredietary supplementation. These diets and conditions have in common alack of essential micronutrients such as metals (K, Ca, Fe, Zn, Mn, Se)and ions (PO₄) necessary for optimal nutrition. Diets rich in phyticacid exacerbate this problem and may also lead to both chronic and acuteconditions that result from either voluntary or economically enforceddependence on diets rich in high phytic acid foods.

For example, individuals following various low carbohydrate (“low carb”)diets are often plagued with muscle, e.g., leg muscle, cramps. Typicaladvice for this is to add additional potassium, calcium and othernutrients to their diet. This invention provides compositions fordietary supplementation, dietary aids and supplements and methods fordiet supplementation to enhance otherwise compromised nutrition via themobilization of macro and micronutrients using phytase supplementationto the diet (including use of any phytase, or, a phytase of theinvention).

In one aspect of the invention, the use of a phytase (e.g., use of anyphytase, or, a phytase of the invention) is optimized to demonstratethermolabile or pH-stability profiles that will make it suitable foraddition directly to the food and supplement process and/or demonstrateenhanced stability and activity in the human or animal gastro intestinaltract.

The invention also provides novel dietary supplements and additives, andmethods of using them, comprising phytases, e.g., any phytase, or, aphytase of the invention, for individuals undergoing mineralsupplementation. Mineral supplementation for people on foods with highphytic acid content may actually exacerbate problems with nutrientavailability. Literature references suggest that complexes of phyticacid, calcium and zinc are much more insoluble that complexes of phyticacid and calcium. People often take multi mineral supplements. Theaddition of phytase to a scheme devised to combine mineral supplementsin the presense of high phytic acid foods could make these supplementsmuch more effective.

In alternative aspects, the compositions and methods of the invention(comprising any phytase, or, a phytase of the invention) are used assupplements or additives to

-   -   Weight-loss programs which limit intake of particular food        groups, vegetarian, macrobiotic or vegan diets which limit or        preclude intake of meats, nightshade vegetables, breads, etc and        other diets which focus on intake of nuts,    -   Specific supplement for individuals on low carb diets rich in        high phytic acid foods to ease physiological symptoms based on        reduced mineral uptake,    -   Athletic training regimens which seek to enhance performance        through dietary intake, including military training regimens,    -   Hospital diets tailored to specific needs of patients        compromised in uptake or restricted to food groups    -   Micronutrient-poor cereal and legume diets in the developing        world,    -   School lunch programs.

The invention also provides kits comprising compositions of theinvention (comprising any phytase, or, a phytase of the invention) andinstructions on incorporating the composition or method of the inventioninto these diets. The kits can comprise any packaging, labeling, productinserts and the like.

In one aspect, the invention provides a natural phytase or an optimizedphytase of the invention, formulated for or optimized for (e.g.,sequence optimized for) production, processing or passage thru human oranimal system, e.g., digestive tract. The phytase enzyme can beoptimized using alternative formulations.

Alternatively, a phytase enzyme of the invention, or, any phytase, canbe optimized by engineering of its sequence, e.g., using for example,directed evolution, error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, ligation reassembly,GSSM™ and any combination thereof, to retain activity during processing,ingestion and in the human gut.

The compositions (e.g., dietary formulations comprising any phytaseenzyme, or a phytase enzyme of the invention) can be delivered in anumber of ways to provide dietary efficacy. For example, the inventionprovides compositions (e.g., dietary formulations or additivescomprising any phytase enzyme, or a phytase enzyme of the invention) andmethods comprising use of:

-   -   In packaged food supplements such as chewable tablets or        nutritional bars,    -   As a lyophilized product available for hydration prior to        ingestion,    -   Co-packaged with dietary products, eg., processed soy product or        sold as a formulation with soybean protein hydrolysate and other        processing fractions from whole foods that are sold as        ingredients to the processed food industry,    -   In commercial baked goods,    -   Spray-on to breakfast cereals,    -   Spray-administered (e.g., nasal spray) formulations,    -   As a transgenic product expressed in indigenous crops, ie.,        cereals and legumes (e.g., as a transgenic product of a        microorganism, such as a bacterium)    -   As a transgenic organism, e.g., a microorganism; for example, a        human or animal is fed a bacteria or other microorganism capable        of making (and, in an alternative embodiment, secreting) a        recombinant phytase, such as a phytase of the invention, after        ingestion or implantation, e.g., into the gut of the human or        animal.

Phytase-containing products and methods of the invention can be brandedas nutrient enhanced, nutrient compatible or otherwise noted for anability to enhance nutrient performance and relieve various symptomsassociated with nutrient deficiency.

Phytase-containing products and methods of the invention are used tomitigate the anti-nutritive effects of phytate, which chelates importantdietary minerals such as zinc, copper, iron, magnesium, tin, andcalcium. According, phytase-containing products and methods of theinvention are used as dietary supplements to prevent the precipitationof metal-binding enzymes and proteins in ingested foods. In one aspect,the phytase-containing products and methods of the invention are used tomitigate the anti-nutritive effects of phytate in human diets, inparticular those rich in legumes and cereals, to increase mineralbioavailability. In one aspect, a phytase in a dietary supplement of theinvention catalyzes the partial or complete hydrolytic removal oforthophosphate from a phytate, where complete hydrolysis of phytateresults in the production of 1 molecule of inositol and 6 molecules ofinorganic phosphate.

Phytase-containing products and methods of the invention are applicableto the diet of humans and numerous animals, including fowl and fish. Forexample, phytase-containing dietary supplement products and dietarysupplement methods of the invention can be practiced with commerciallysignificant species, e.g., pigs, cattle, sheep, goats, laboratoryrodents (rats, mice, hamsters and gerbils), fur-bearing animals such asmink and fox, and zoo animals such as monkeys and apes, as well asdomestic mammals such as cats and dogs. Typical commercially significantavian species include chickens, turkeys, ducks, geese, pheasants, emu,ostrich, loons, kiwi, doves, parrots, cockatiel, cockatoo, canaries,penguins, flamingoes, and quail. Commercially farmed fish such as troutwould also benefit from the dietary aids disclosed herein. Other fishthat can benefit include, for example, fish (especially in an aquariumor aquaculture environment, e.g., tropical fish), goldfish and otherornamental carp, catfish, trout, salmon, shark, ray, flounder, sole,tilapia, medaka, guppy, molly, platyfish, swordtail, zebrafish, andloach.

Phytase-containing products and methods of the invention are also usedin various agars, gels, medias, and solutions used in tissue and/or cellculturing. Inconsistent soy hydrolysates can be a problem encounteredwhen using tissue and/or cell culturing. In one aspect,phytase-containing products and methods of the invention are used ascell culture media additives or as treatments to, e.g., increase cellculture yield and performance consistency. In one aspect, the inventionprovides hydrolysate for cell culturing comprising phytases, e.g.,phytases of the invention.

In one aspect, to provide a consistent product, the invention providemethods for making hydrolysates, supplements or other additives for cellculturing comprising phytases by using phytase biomarkers. For example,the method would comprise “scoring” or “marking” several molecules ofphytase in batches of hydrolysate, supplement or other additive, andthen blending the batches in the hydrolysate, supplement or otheradditive to achieve a consistent biomarker pattern. In one aspect,culture performance with each batch is measured in a mini-bioreactor(s)and performance with each biomarker and batch is correlated. In oneaspect, a blend is made to generate a higher performance product that isconsistent or better than average. In one aspect, thioredoxin (TRX) isadded to increase the bioavailability of many proteins by eliminatingsecondary structure caused by disulfide bonds. In one aspect, proteasesare also added to the hydrolysates, supplements or additives of theinvention. The proteases can be “scored” or quality controlled withother biomarkers (as with phytase, as discussed above) to direct theblending process.

In one aspect, the invention provides methods for adding phytases tograins to provide a consistent product using a biomarker “scoring” orquality control process analogous to that described above for thehydrolysates, supplements or additives of the invention.

Enzyme Enhanced Diets for Increased Warfighter Efficiency and Morale

In one aspect, the invention provides novel dietary supplements andadditives and methods for diet supplementation comprising phytases,e.g., any phytase, or, a phytase of the invention, for enzyme-enhanceddiets for increased warfighter efficiency and morale. In one aspect,these dietary supplement compositions of the invention work, in situ, toenhance energy, stamina and morale in a stable, easily usable anddesirable format while limiting food waste.

In one aspect, these dietary supplement compositions and methods of theinvention address the military operational challenge comprisingefficient delivery of nutrients and the associated health, morale andoperational effectiveness of soldiers. The invention provides enzymesoptimized to function efficiently in the human gut. These enzymes canenhance extraction of nutrients and generation of energy as well asprolong maintenance of nutritional sufficiency and individual satiety.

In addition to phytase, other enzymes, e.g., amylases, xylanases,proteases, lipases, are used to practice the dietary supplementcompositions and methods of the invention. In one aspect, the inventionprovides formulations, food supplements, foods, self-contained mealReady-to-Eat units (MREs), drinks, hydrating agents and the like,comprising phytase, e.g., a phytase of the invention, and anotherenzyme, e.g., amylases, xylanases, proteases, lipases or a combinationthereof. When ingested with food, these enzymes have been shown toenhance the release of critical nutrients, e.g., phosphorus, essentialmetals and ions, amino acids, and sugars. Furthermore, co-ingestion ofthese enzymes increases gastrointestinal mechanics and absorption bydepolymerizing plant-derived cellulose, hemicellulose and starch. Thiswhite paper proposes the development of these enzymes as supplements tomilitary diets to provide enhanced nutrient utilization for warfighter.

In one aspect, the food supplement of the invention causes the releaseof essential phosphate from normally anti-nutritive, plant-derivedphytate to increase food energy yield and bone CaPO₄ deposition. In oneaspect, phytases and other potential nutritional supplement enzymes canwithstand gut pH and endogenous protease activities.

In one aspect, the invention provides enzyme supplements to rations,drinks, foods, MREs, hydrating agents and the like to significantlyimprove nutritional value, digestibility and energy content of militarymeals (or any meal, including general consumer meal and dietsupplementation products) served to warfighters in training, battle orany stressful situation. The supplement can be formulated for ease ofuse and personal transport (in or with MREs, hydrating agents, etc). Inone aspect, the enzyme supplement will not compromise food appearance,taste and/or consistency. In one aspect, the product improve health andincreases the stamina of warfighters.

In alternative aspects, the enzyme supplement is delivered in a numberof ways to provide dietary efficacy; for example, the invention providesphytases, including phytases of the invention, and in some aspects,additional enzymes, in:

-   -   Packaged food or drink supplements such as MREs, rations,        survival kits, hydrating agents, chewable tablets or nutritional        bars;    -   As a lyophilized product (e.g., a powder) available for        hydration prior to ingestion;    -   Co-packaged with dietary products, foods, drinks, e.g.,        processed soy product or a formulation with soybean protein        hydrolysate and other processing fractions from whole foods that        are sold as ingredients to the processed food industry;    -   In baked goods;    -   Spray-on to cereals;    -   Formulations such as tablets, geltabs, capsules, sprays and the        like.

In one aspect, the compositions and methods of the invention providenutritional supplementation that rapidly releases calories and macro-and micronutrients from ingested meals. In one aspect, the compositionsand methods of the invention provides energy and body strength toindividuals in stressful situations, e.g., involving hyperexertion anddiscontinuous periods of depravation. In one aspect, the compositionsand methods of the invention provide enzymes optimized and formulated towork effectively in human gut while maintaining stability, shelf lifeand transportability in a desired environment, e.g., a military setting.

In one aspect, the compositions and methods of the invention provideformulations for increased taste characteristics, dissolvability,chewability and personal transport efficiency of the product. In oneaspect, the compositions and methods of the invention further compriseother components, such as potassium, glucose, CaCl₂. CaCl₂ in theformulation can combine with released phosphate and, in turn, enhancebone deposition and weight gain. In one aspect, the compositions andmethods of the invention further comprise formulations of other enzymes,such as proteases, cellulases, hemicellulases, for protein, celluloseand hemicellulose digestion, respectively. These enzymes can improveprotein and starch availability and further increase iron absorptionfrom many iron-rich foods.

In one aspect, the compositions and methods of the invention furthercomprise enzymes for hydrolyzing foods derived from plant material,which is rich in the glucose and xylose-based polymers, cellulose,hemicellulose and starch, as well as in the amino acid polymer, protein.In one aspect, the compositions and, methods of the invention facilitatehydrolysis of polymeric materials in foods; i.e., to facilitate completedigestion polymers to monomers, e.g., polysaccharides to monomericsugars, or proteins to amino acid moieties. Thus, in this aspect, thecompositions and methods of the invention allow a food, drink or rationto realize its full caloric and nutritional value. In one aspect, enzymesupplementation comprises use of stabile enzymes, e.g., hydrolases ofvarious kinds, cellulases, hemicellulases, amylases, lipases, amidases,proteases and other enzymes. In one aspect, enzymes used in thecompositions and methods of the invention can withstand ambient gutconditions, i.e., stability at low pH and in the presence of gastricproteases.

Industrial Uses of Phytases

In addition to those described above, the invention provides novelindustrial uses for phytases, inlcuding use of the novel phytases of theinvention.

In one aspect, the invention provides compositions comprising phytases(including the phytases of the invention) for addition to waste ormanure piles to convert “environmental” phytic acid. In one aspect, thisserves the purposes of reducing pollution and increasing nutrientavailability. The invention also provides compositions and methods foradding a phytase to soil, natural or artificial bodies of water (e.g.,lakes, ponds, wells, manure ponds, and the like), municipal sewage, anysewage effluent, and the like. As described above, the inventionprovides compositions and methods for reducing phytate levels in wasteor sewage, e.g., an animal manure, wherein the animal is fed a dietaryaid containing an effective amount of a phytase, e.g., a phytase of theinvention. An exemplary application of the compositions and methods ofthe invention is to reduce the phosphate pollution in the environment.Thus, the compositions and methods of the invention can be used in anyapplication that reduces pollution by degrading phytic acids.

In one aspect, the invention provides compositions comprising phytases(including the phytases of the invention) and methods for farmingapplications or other plant growth applications, e.g., adding phytasesto fertilizers or plant food additives (e.g., MIRACLEGROW™) for plants,e.g., house plants. In using the compositions and methods of theinvention for farming applications, users include organic farmers. Thecompositions and methods of the invention can be used for addingphytases to any soil deficient in phosphorous or needing supplementaryphosphorous for a particular crop or application. Because phosphorousrelease helps plants grow, compositions and methods of the invention canbe used for adding phytases to anything that has algae or plant materialin it.

In one aspect, the invention provides compositions comprising phytases(including the phytases of the invention) and methods for cosmeticapplications, e.g., shampoos, lotions or soaps containing plantproducts.

In one aspect, the invention provides compositions comprising phytases(including the phytases of the invention) and methods for immobilizingthe phytase. In one aspect, the immobilized phytase acts as a controlledrelease mechanism. For example, in one aspect, the invention providescontrol released (time release) formulations of phytases for applicationto soil, e.g., clay, to house plants, etc. In one aspect, the phytasesare immobilized to beads, e.g., polysorb beads. These beads can bedelivered to soil, e.g., for agricultural or house plants. In anotheraspect, control released (time release) formulations of phytases of theinvention are used in dietary supplements and additives.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples. While the procedures described in the examplesare typical of those that can be used to carry out certain aspects ofthe invention, other procedures known to those skilled in the art canalso be used.

EXAMPLES Example 1 Isolation, Bacterial Expression, and Purification ofPhytase

E. coli B genomic DNA was obtained from Sigma (Catalog # D-2001), St.Louis, N.J.

The following primers were used to PCk amplify the gene directly fromthe genomic DNA: (SEQ ID NO: 3) 5′ primergtttctgaattcaaggaggaatttaaATGAAAGCGATCTTAATCCCATT; and (SEQ ID NO: 4) 3′primer gtttctggatccTTACAAACTGCACGCCGGTAT.

Pfu polymerase in the PCR reaction, and amplification was performedaccording to manufacturers protocol (Stratagene Cloning Systems, Inc.,La Jolla, Calif.).

PCR product was purified and purified product and pQE60 vector (Qiagen)were both digested with EcoRI and BglII restriction endonucleases (NewEngland Biolabs) according to manufacturers protocols. Overnightligations were performed using standard protocols to yield pQE60.

The amplified sequences were inserted in frame with the sequenceencoding for the RBS. The ligation mixture was then used to transformthe E. coli strain M15/pREP4 (Qiagen, Inc.) by electroporation. MI5/pREP4 contains multiple copies of the plasmid pREP4, which expressesthe lacI repressor and also confers kanamycin resistance (Kan^(r)).Plasmid DNA was isolated and confirmed by restriction analysis. Clonescontaining the desired constructs were grown overnight (O/N) in liquidculture in LB media supplemented with both Amp (100 ug/ml) and Kan (25ug/ml). The O/N culture was used to inoculate a large culture at a ratioof 1: 100 to 1:250. The cells were grown to an optical density 600(O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalactopyranoside”) was then added to a final concentration of 1 mM. IPTGinduces by inactivating the lacI repressor, clearing the P/O leading toincreased gene expression. Cells were grown an extra 3 to 4 hours. Cellswere then harvested by centrifugation.

The primer sequences set out above may also be employed to isolate thetarget gene from the deposited material by hybridization techniquesdescribed above.

This invention also provides for the isolation and use of phytasemolecules (nucleic acids and phytase enzymes encoded thereby) from allother strains of E. coli (whether virulent or non-virulent, includingK12, W, C), as well as all bacteria. These include all known species andstrains belonging to:

-   Thermotogales-   Green Nonsulfur Bacteria-   Cyanobacteria & chloroplasts-   Low G+C Gram-Positive Bacteria-   Fusobacteria-   High G+C Gram-Positive Bacteria-   Gytophaga/Flexibacter/Bacteroides group-   Fibrobacteria-   Spriochaetes-   Planctomyces/Chlamydia group-   Purple bacteria (Proteobacteria), including the following    subdivisions:    -   Delta & Epsilon, including:        -   Desulfuromonas acetoxidans        -   Desulfosarcina variabilis        -   Bdellovibrio stolpii        -   Nannocystis exedens        -   Stigmatella aurantiaca        -   Myxococcus xanthus        -   Desulfovibrio desulfuricans        -   Thiovulum sp.        -   Campylobacter jejuni        -   Wolinella succinogenes        -   Helicobacter pylori    -   Alpha, including:        -   Methylobacterium extorquens        -   Beijerinckia indica        -   Hyphomicrobium vulgare        -   Rhodomicrobium vannieli        -   Agrobacterium tumefaciens        -   Brucella abortus        -   Rochalimaea quintana        -   Rhodopseudomonas marina subsp. agilis        -   Zea mays-mitochondrion        -   Rickettsia rickettsii        -   Ehrlichia risticii        -   Wolbachia pipientis        -   Anaplasma marginale        -   Erythrobacter longus        -   Rhodospirillum salexigens        -   Rhodobacter capsulatus        -   Azospirillum lipoferum        -   Rhodospirillum rubrum    -   Gamma, including:        -   Ectothiorhodospira shaposhnikovii        -   Chromatium vinosum        -   Methylomonas methanica        -   Cardiobacterium hominis        -   Xanthomonas maltophilia        -   Coxiella burnetii        -   Legionella pneumophila subsp. pneumophila        -   Oceanospirillum linum        -   Acinetobacter calcoaceticus        -   Pseudomonas aeruginosa        -   Haemophilus influenzae        -   Vibrio parahaemolyticus        -   Proteus vulgaris        -   Erwinia carotovora        -   Escherichia coli, including:    -   Beta, including:        -   Eikenella corrodens        -   Neisseria gonorrhoeae        -   Vitreoscilla stercoraria        -   Chromobacterium violaceum        -   Alcaligenes faecalis        -   Rubrivivax gelatinosus        -   Pseudomonas testosteroni        -   Nitrosomonas europae        -   Spirillum volutans

Substances that alter the digestive flora environment of the consumingorganism to enhance growth rates. As such, there are many problematicburdens—related to nutrition, ex vivo processing steps, health andmedicine, environmental conservation. Such phytase molecules can beisolated from these bacteria by know methods, including libraryscreening methods, e.g. expression screening, hybridization methods, PCR(e.g. see Sammbrook, 1989).

Example 2 Thermal Tolerance Assay

The wild type appA from E. coli (strain K12) and a mutagenized versiondesignated 819PH59 (SEQ ID NO:9 and 10) were expressed in E. coli andpurified to homogeneity. In the thermal tolerance assay, 100 uL of 0.01mg/mL of protein in 100 mM MOPS/pH 7.0 was heated to the indicatedincubation temperature for 5 minutes in an RJ research thermocycler.Upon completion of the 5 minutes at the incubation temperature, thesamples were cooled to 4° C. and incubated on ice. An activity assay wasrun using 40 uL of the enzyme solution in 1.5 mL of 100 mM NaOAc/4 mMphytate/pH 4.5 at 37° C. Aliquots of 60 uL were withdrawn at 2 minuteintervals and added to 60 uL of the color developer/Stop solution of theTNO assay. Clearly, the modified enzyme, SEQ ID NO:10, containing 8amino acid changes, is tolerant to temperatures greater than thewild-type enzyme. See FIG. 3.

Example 3 Stability of Phytase Enzyme in Simulated DigestibilityConditions

The percent residual activities (based on initial rates) of the in vitrodigested E. coli K12 and the nonglycosylated 819pH59 phytase wereplotted verses time. A standard concentration of simulated gastric fluidcontaining 2 mg/ml NaCl, 6 M HCl and 3.2 mg/mL pepsin was prepared asdescribed. The pH of the solution was about 1.4 and was not adjusted.The in vitro digestibility assay was performed by adding 1:4 (vol:vol)of phytase to digestion solution and immediately incubating at 37° C. toinitiate the digestion reaction. Aliquots of the digestion reactionmixture were removed at various time intervals and assayed for residualphytase activity using the TNO assay. Each of the assays was performedat least twice. An exponential curve with the equation y=Ae−kt was fitto the data. The half lives of the proteins were determined using theequation t 1/2=In 2/k. The half-life of the E. coli K12 phytase was only2.7±0.2 minutes while the nonglycosylated 819pH59 phytase had ahalf-life of 8.4±1.1 minutes. Therefore, the mutations in the wildtypeE. coli K12 phytase enhanced the stability of the enzyme under simulatedin vitro digestibility conditions. See FIG. 4.

Example 4 Expression Host Comparisons

The GSSM™ DNA construct from 819PH59 was inserted into E. coli, P.pastoris, and S. pombe for expression. The expressed proteins werepurified to homogeneity. In the thermal tolerance assay, 100 uL of 0.01mg/mL of protein in 100 mM MOPS, pH 7.0 was heated to the indicatedincubation temperature for 5 minutes in an RJ research thermocycler.Upon completion of the 5 minutes at the incubation temperature, thesamples were cooled to 4° C. and incubated on ice. An activity assay wasrun using 40 uL of the enzyme solution in 1.46 mL of 100 mM NaOAc/4 mMphytate/pH 4.5 at 37° C. Aliquots of 60 uL were withdrawn at 2 minuteintervals and added to 60 uL of the color developer/Stop solution of theTNO assay. See FIG. 5.

Example 5

The percent residual activities (based on initial rates) of the in vitrodigested 819pH59 phytase expressed in various expression hosts wereplotted verses time. The 819pH59 phytase was expressed in E. coli(nonglycosylated), as well as in S. pombe and P. pastoris(glycosylated). A standard concentration of simulated gastric fluidcontaining 2 mg/ml NaCl, 6 M HCl and 3.2 mg/mL pepsin was prepared asdescribed in the S.O.P. The pH of the solution was about 1.4 and was notadjusted. The in vitro digestibility assay was performed by adding 1:4(vol:vol) of phytase to digestion solution and immediately incubating at37° C. to initiate the digestion reaction. Aliquots of the digestionreaction mixture were removed at various time intervals and assayed forresidual phytase activity using the TNO assay. Each of the assays wasperformed in triplicate. An exponential curve with the equationy=Ae^(−kt) was fit to the data. The half lives of the proteins weredetermined using the equation t_(1/2)=1n 2/k. The half-life of thenonglycosylated 819pH59 phytase expressed in E. coli was 8.4±1.1 minuteswhile the glycosylated 819pH59 phytase expressed in S. pombe had ahalf-life of 10.4±0.9 minutes and the same phytase expressed in P.pastoris had a half-life of 29.2±6.7 mins. Therefore, the glycosylationof the 819pH59 phytase enhanced the stability of the enzyme undersimulated in vitro digestibility conditions. See FIG. 6.

Literature Cited

(The teachings of all references cited in this application are herebyincorporated by reference in their entirety unless otherwise indicated.)

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Example 6

The following example describes an exemplary assay that can be used todetermine if a polypeptide has phytase activity and is within the scopeof the invention.

-   -   pH and temperature profile and stability data were measured for        the exemplary phytase having a sequence as set forth in SEQ ID        NO:2. FIGS. 2A and 2B show the pH and temperature profile and        stability data, respectively, for an exemplary phytase enzyme of        the present invention (having a sequence as set forth in SEQ ID        NO:2). The assay used for these analyses is the following for        the detection of phytase activity: Phytase activity is measured        by incubating 10 μl of the enzyme preparation with 65 μl of 2 mM        sodium phytate in 100 mM Tris maleate buffer pH 5.2,        supplemented with 1 mM CaCl₂ for 30 minutes at 42° C. After        incubation the reaction is stopped by adding 75 μl of 5%        trichloroacetic acid. Phosphate released was measured against        phosphate standard spectrophotometrically at 700 nm after adding        150 μl of the color reagent (4 volumes of 1.5% ammonium        molybdate in 5.5% sulfuric acid and 1 volume of 2.7% ferrous        sulfate; Shimizu, M., 1992; Biosci. Biotech. Biochem        56:1266-1269). OD at 700 nm is indicated on the Y-axis of the        graphs in FIG. 2. Temperature or pH is indicated on the X-axis        of the graphs.

Example 7

The following example describes an exemplary assay that can be used todetermine if a polypeptide has phytase activity and is within the scopeof the invention. This exemplary assay, also described by Engelen (1994)J. of AOAC International 77(3):760-764 (see also Nagashima (1999) ApplEnviron Microbiol. 65(10):4682-4684), can be used as a simple and rapiddetermination as to whether a polypeptide has phytase activity underacidic conditions, e.g., at pH 5.5. The method is based on thedetermination of inorganic orthophosphate released on hydrolysis ofsodium phytate at pH 5.5.

Weighed samples were diluted in duplicate with buffer (adjusted to pH5.5 with acetic acid) and placed in waterbath at 37° C. Substratesolution (sodium phytate from rice adjusted to pH 5.5) added, samplesmixed, and at 65 minutes incubation terminated with color-stop mix(ammonium heptamolybdate and ammonium vanadate). Tubes with samplecentrifuged for 5 min. and absorbance measured at 415 nm withspectrophotometer. Corrected absorbance difference calculated bysubtracting absorbance blank from that of corresponding sample. Enzymeactivity is expressed in activity units (FTU), where 1 FTU is the amountof enzyme that liberates 1 mmol inorganic orthophosphate per min underthe test conditions (pH 5.5, 37° C.) See Engelen (1994) supra.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described. It is to be understood that, while theinvention has been described with reference to the above detaileddescription, the foregoing description is intended to illustrate, butnot to limit, the scope of the invention. Other aspects, advantages, andmodifications of the invention are within the scope of the followingclaims. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety.

Although the invention has been described with reference to thepresently preferred embodiments, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1. A formulation comprising at least one polypeptide having phytaseactivity, wherein the polypeptide comprises: (a) a polypeptide encodedby a nucleic acid comprising a nucleotide sequence as set forth in SEQID NO:7 and wherein nucleotide 389 is G; 390 is A; 437 is T; 438 is G;439 is G; 470 is C; 472 is T; 476 is T; 477 is G; 478 is T; 689 is G;690 is A; 691 is G; 728 is T; 729 is A; 730 is T; 863 is T; 864 is G;or, 1016 is G; or, any combination thereof, wherein the polynucleotideencodes a phytase; (b) a polypeptide encoded by a nucleic acidcomprising a nucleotide sequence as set forth in SEQ ID NO:7 and whereinnucleotide 389 is G; 390 is A; 437 is T; 438 is G; 439 is G; 470 is C;472 is T; 476 is T; 477 is G; 478 is T; 689 is G; 690 is A; 691 is G;728 is T; 729 is A; 730 is T; 863 is T; 864 is G; and 1016 is G; (c) apolypeptide having an amino acid sequence as set forth in SEQ ID NO:8and having one or more amino acid modifications selected from W68E,Q84W, A95P, K97C, S168E, R181Y, N226C, Y277D or any combination thereof,wherein the polypeptide has phytase activity; (d) a polypeptide havingan amino acid sequence as set forth in SEQ ID NO:8 and having the aminoacid modifications W68E, Q84W, A95P, K97C, S168E, R181 Y, N226C, Y277D,wherein the polypeptide has phytase activity; (e) a polypeptide encodedby a nucleic acid comprising a nucleotide sequence as set forth in SEQID NO:1; (f) a polypeptide having an amino acid sequence as set forth inSEQ ID NO:2; or, (g) a combination of (a), (b), (c), (d), (e) or (f). 2.The formulation of claim 1, wherein the formulation is a dietarysupplement.
 3. The formulation of claim 1, further comprising at leastone vitamin, at least one additional enzyme, at least one mineral ormetal, or at least one herb or plant extract, at least one amino acid oramino acid derivative, or any combination thereof.
 4. The formulation ofclaim 3, wherein the mineral or metal is selected from the groupconsisting of aluminum, antimony, barium, beryllium, bismuth, boron,bromide, bromine, cadmium, calcium, cerium, cesium, chloride, chromium,cobalt, copper, dysprosium, erbium, europium, fluoride, fluorine,gadolinium, gallium, germanium, gold, hafnium, holmium, indium, iodine,iridium, iron, lanthanum, lithium, lutetium, magnesium, manganese,molybdenum, neodymium, nickel, niobium, osmium, palladium, phosphorous,platinum, potassium, praseodymium, promethium, rhenium, rhodium,rubidium, ruthenium, samarium, scandium, selenium, silicon, silver,sodium, strontium, sulfur, tantalum, tellurium, terbium, thorium,thulium, tin, titanium, tungsten, vanadium, xinconium, ytterbium,yttrium, zinc, zirconium and any combination thereof.
 5. The formulationof claim 1, further comprising at least one composition selected fromthe group consisting of diatomaceous earth, charcoal, choline, inositol,biotin, PABA, Alpha-Lipoic Acid, a carotenoid, beta carotene, coenzymeQ10, chondroitin, melatonin, lecithin, brewer's yeast and a combinationthereof.
 6. The formulation of claim 3, wherein the one herb or plantextract is selected from the group consisting of alfalfa, a ginseng,American ginseng, Asian red ginseng, Asian white ginseng, Siberianginseng, Brazilian ginseng, astragalus, bilberry, black cohosh, cascarasagrada, cat's claw, cayenne, dong quai, echinacea, eucalyptus,feverfew, garlic, ginkgo biloba, goldenseal, gotu kola, horsetail, maca,a mushroom, Maitake mushroom, Reishi mushroom, Shuitake mushroom,leuzea, rhodiola, milk thistle, noni, pau d'arco, papaya, pygeum, sawpalmetto, schizandra, senna, suma, wild yam, willow, yucca, wheat grass,barley grass, parsley, broccoli, acerola cherries, aloe vera, quercitin,pine bark, grape seed, green tea, red wine, grapefruit extract, ginger,oat straw, sarsaparilla, an oil, walnut oil, safflower oil, soybean oil,peanut oil, a fish oil, salmon oil, evening primrose oil, borage oil,bee pollen, bee propolis, royal jelly, a bran, oat bran, wheat bran, afiber, soy, psyllium, apple pectin, a protein, egg protein, milkprotein, soy protein, rice protein, whey, algae, spirulina, Chlorella,dulse, kelp, D. salina and a combination thereof.
 7. The formulation ofclaim 3, wherein the probiotic is selected from the group consisting ofa Lactobacillus species, L. acidophilus, L. bifidus, L. sporogenes, L.casei, L. rhamnosus, L. plantarum, S. thermophilus, a Bifidobacteriumspecies, an Escherichia, an Enterococcus, a Bacillus and a Saccharomycesspecies.
 8. The formulation of claim 3, wherein the additional enzyme isselected from the group consisting of a phytase, an amylase, abromelain, a cellulase, a chymopapain, a diastase, a glucoamylase, ahemicellulase, a hyaluronidase, an invertase, a lactase, a lipase, amaltase, a pancreatin, a papain, a pectinase, a pepsin, a plasmin, aprotease, a rennin and any combination thereof.
 9. The formulation ofclaim 3, wherein the vitamin is selected from the group consisting ofvitamin B, Thiamine (Vitamin B1), Riboflavin (Vitamin B2), Nicotinicacid (Niacin, Vitamin B3), Pantothenic acid (Vitamin B5), Pyridoxine(Vitamin B6), B7, Folic acid (Vitamin B9), Cyanocobalamin (Vitamin B12),vitamin C, a vitamin D, vitamin D1, vitamin D2, vitamin D3, vitamin E, avitamin K, vitamin K1, vitamin K2, vitamin G, vitamin H, vitamin P andany combination thereof.
 10. The formulation of claim 3, wherein theamino acid or amino acid derivative is selected from the groupconsisting of Isoleucine, Leucine, Lysine, Phenylalanine, Threonine,Tryptophan, Valine, Methionine, Cysteine, Alanine, Arginine, AsparticAcid, Glutamic Acid, Glycine, Histidine, Proline, Serine, Asparagine,Glutamine, Tyrosine, taurine, glucosamine and any combination thereof.11. The formulation of claim 1, comprising vitamin D3 or calcium orboth.
 12. The formulation of claim 1, further comprising potassium,glucose, CaCl₂ or a combination thereof.
 13. The formulation of claim 1,further comprising at least one enzyme selected from the groupconsisting of α-galactosidases, β-galactosidases, lactases, phytases,β-glucanases, endo-β-1,4-glucanases and endo-β-1,3(4)-glucanases,cellulases, xylosidases, galactanases, arabinogalactanendo-1,4-β-galactosidases and arabinogalactan endo-1,3-β-galactosidases,endoglucanases, endo-1,2-β-glucanase, endo-1,3-α-glucanase,endo-1,3-β-glucanase, pectin degrading enzymes, pectinases,pectinesterases, pectin lyases, polygalacturonases, arabinanases,rhamnogalacturonases, rhamnogalacturonan acetyl esterases,rhamnogalacturonan-α-rhamnosidase, pectate lyases, α-galacturonisidases,mannanases, β-mannosidases, mannan acetyl esterases, xylan acetylesterases, proteases, xylanases, arabinoxylanases, lipases,phospholipases and cutinases.
 14. The formulation of claim 1, whereinthe formulation comprises a powder, a tablet, a concentrate, a geltab, acapsule, a spray, an aerosol, a lotion, an adhesive patch or a drink.15. A pharmaceutical composition comprising at least one polypeptidehaving phytase activity and a pharmaceutically acceptable excipient,wherein the polypeptide comprises: (a) a polypeptide encoded by anucleic acid comprising a nucleotide sequence as set forth in SEQ IDNO:7 and wherein nucleotide 389 is G; 390 is A; 437 is T; 438 is G; 439is G; 470 is C; 472 is T; 476 is T; 477 is G; 478 is T; 689 is G; 690 isA; 691 is G; 728 is T; 729 is A; 730 is T; 863 is T; 864 is G; or, 1016is G; or, any combination thereof, wherein the polynucleotide encodes aphytase; (b) a polypeptide encoded by a nucleic acid comprising anucleotide sequence as set forth in SEQ ID NO:7 and wherein nucleotide389 is G; 390 is A; 437 is T; 438 is G; 439 is G; 470 is C; 472 is T;476 is T; 477 is G; 478 is T; 689 is G; 690 is A; 691 is G; 728 is T;729 is A; 730 is T; 863 is T; 864 is G; and 1016 is G; (c) a polypeptidehaving an amino acid sequence as set forth in SEQ ID NO:8 and having oneor more amino acid modifications selected from W68E, Q84W, A95P, K97C,S168E, R181Y, N226C, Y277D or any combination thereof, wherein thepolypeptide has phytase activity; (d) a polypeptide having an amino acidsequence as set forth in SEQ ID NO:8 and having the amino acidmodifications W68E, Q84W, A95P, K97C, S168E, R181 Y, N226C, Y277D,wherein the polypeptide has phytase activity; (e) a polypeptide encodedby a nucleic acid comprising a nucleotide sequence as set forth in SEQID NO:1; (f) a polypeptide having an amino acid sequence as set forth inSEQ ID NO:2; or, (g) a combination of (a), (b), (c), (d), (e) or (f).16. The pharmaceutical composition of claim 15 formulated for oraldelivery.
 17. The pharmaceutical composition of claim 15 formulated as apill, a tablet, a capsule, a spray, an aerosol or a powder.
 18. A kitcomprising a formulation as set forth in claim 1, or a pharmaceuticalcomposition as set forth in claim 7, and instructions on using theformulation or the pharmaceutical composition.
 19. An immobilizedphytase comprising: (a) a polypeptide encoded by a nucleic acidcomprising a nucleotide sequence as set forth in SEQ ID NO:7 and whereinnucleotide 389 is G; 390 is A; 437 is T; 438 is G; 439 is G; 470 is C;472 is T; 476 is T; 477 is G; 478 is T; 689 is G; 690 is A; 691 is G;728 is T; 729 is A; 730 is T; 863 is T; 864 is G; or, 1016 is G; or, anycombination thereof, wherein the polynucleotide encodes a phytase; (b) apolypeptide encoded by a nucleic acid comprising a nucleotide sequenceas set forth in SEQ ID NO:7 and wherein nucleotide 389 is G; 390 is A;437 is T; 438 is G; 439 is G; 470 is C; 472 is T; 476 is T; 477 is G;478 is T; 689 is G; 690 is A; 691 is G; 728 is T; 729 is A; 730 is T;863 is T; 864 is G; and 1016 is G; (c) a polypeptide having an aminoacid sequence as set forth in SEQ ID NO:8 and having one or more aminoacid modifications selected from W68E, Q84W, A95P, K97C, S 168E, R181 Y,N226C, Y277D or any combination thereof, wherein the polypeptide hasphytase activity; (d) a polypeptide having an amino acid sequence as setforth in SEQ ID NO:8 and having the amino acid modifications W68E, Q84W,A95P, K97C, S168E, R181 Y, N226C, Y277D, wherein the polypeptide hasphytase activity; (e) a polypeptide encoded by a nucleic acid comprisinga nucleotide sequence as set forth in SEQ ID NO:1; (f) a polypeptidehaving an amino acid sequence as set forth in SEQ ID NO:2; or, (g) acombination of (a), (b), (c), (d), (e) or (f).
 20. The immobilizedphytase of claim 19, wherein the polypeptide is immobilized to a bead.21. The immobilized phytase of claim 20, wherein the polypeptide isimmobilized to a polysorb bead or a polystyrene bead.
 22. A dietarysupplement comprising the immobilized phytase of claim
 19. 23. Apharmaceutical composition comprising the immobilized phytase of claim19.
 24. A fertilizer or soil additive comprising the immobilized phytaseof claim
 19. 25. A fertilizer or soil additive comprising at least onepolypeptide having phytase activity, wherein the polypeptide comprises:(a) a polypeptide encoded by a nucleic acid comprising a nucleotidesequence as set forth in SEQ ID NO:7 and wherein nucleotide 389 is G;390 is A; 437 is T; 438 is G; 439 is G; 470 is C; 472 is T; 476 is T;477 is G; 478 is T; 689 is G; 690 is A; 691 is G; 728 is T; 729 is A;730 is T; 863 is T; 864 is G; or, 1016 is G; or, any combinationthereof, wherein the polynucleotide encodes a phytase; (b) a polypeptideencoded by a nucleic acid comprising a nucleotide sequence as set forthin SEQ ID NO:7 and wherein nucleotide 389 is G; 390 is A; 437 is T; 438is G; 439 is G; 470 is C; 472 is T; 476 is T; 477 is G; 478 is T; 689 isG; 690 is A; 691 is G; 728 is T; 729 is A; 730 is T; 863 is T; 864 is G;and 1016 is G; (c) a polypeptide having an amino acid sequence as setforth in SEQ ID NO:8 and having one or more amino acid modificationsselected from W68E, Q84W, A95P, K97C, S168E, R181Y, N226C, Y277D or anycombination thereof, wherein the polypeptide has phytase activity; (d) apolypeptide having an amino acid sequence as set forth in SEQ ID NO:8and having the amino acid modifications W68E, Q84W, A95P, K97C, S168E,R181Y, N226C, Y277D, wherein the polypeptide has phytase activity; (e) apolypeptide encoded by a nucleic acid comprising a nucleotide sequenceas set forth in SEQ ID NO:1; (f) a polypeptide having an amino acidsequence as set forth in SEQ ID NO:2; or, (g) a combination of (a), (b),(c), (d), (e) or (f).
 26. A liquid supplement for preventing musclecramps comprising a formulation as set forth in claim
 1. 27. The liquidsupplement of claim 26, further comprising glucose, potassium, sodium orcalcium.
 28. A hydrating agent comprising a formulation as set forth inclaim
 1. 29. The hydrating agent of claim 28, further comprisingglucose, potassium, sodium or calcium.
 30. A tissue culture or cellculture media or cell culture media additive comprising at least onepolypeptide having phytase activity, wherein the polypeptide comprises:(a) a polypeptide encoded by a nucleic acid comprising a nucleotidesequence as set forth in SEQ ID NO:7 and wherein nucleotide 389 is G;390 is A; 437 is T; 438 is G; 439 is G; 470 is C; 472 is T; 476 is T;477 is G; 478 is T; 689 is G; 690 is A; 691 is G; 728 is T; 729 is A;730 is T; 863 is T; 864 is G; or, 1016 is G; or, any combinationthereof, wherein the polynucleotide encodes a phytase; (b) a polypeptideencoded by a nucleic acid comprising a nucleotide sequence as set forthin SEQ ID NO:7 and wherein nucleotide 389 is G; 390 is A; 437 is T; 438is G; 439 is G; 470 is C; 472 is T; 476 is T; 477 is G; 478 is T; 689 isG; 690 is A; 691 is G; 728 is T; 729 is A; 730 is T; 863 is T; 864 is G;and 1016 is G; (c) a polypeptide having an amino acid sequence as setforth in SEQ ID NO:8 and having one or more amino acid modificationsselected from W68E, Q84W, A95P, K97C, S168E, R181Y, N226C, Y277D or anycombination thereof, wherein the polypeptide has phytase activity; (d) apolypeptide having an amino acid sequence as set forth in SEQ ID NO:8and having the amino acid modifications W68E, Q84W, A95P, K97C, S168E,R181 Y, N226C, Y277D, wherein the polypeptide has phytase activity; (e)a polypeptide encoded by a nucleic acid comprising a nucleotide sequenceas set forth in SEQ ID NO:1; (f) a polypeptide having an amino acidsequence as set forth in SEQ ID NO:2; or, (g) a combination of (a), (b),(c), (d), (e) or (f).
 31. A plant food additive comprising at least onepolypeptide having phytase activity, wherein the polypeptide comprises:(a) a polypeptide encoded by a nucleic acid comprising a nucleotidesequence as set forth in SEQ ID NO:7 and wherein nucleotide 389 is G;390 is A; 437 is T; 438 is G; 439 is G; 470 is C; 472 is T; 476 is T;477 is G; 478 is T; 689 is G; 690 is A; 691 is G; 728 is T; 729 is A;730 is T; 863 is T; 864 is G; or, 1016 is G; or, any combinationthereof, wherein the polynucleotide encodes a phytase; (b) a polypeptideencoded by a nucleic acid comprising a nucleotide sequence as set forthin SEQ ID NO:7 and wherein nucleotide 389 is G; 390 is A; 437 is T; 438is G; 439 is G; 470 is C; 472 is T; 476 is T; 477 is G; 478 is T; 689 isG; 690 is A; 691 is G; 728 is T; 729 is A; 730 is T; 863 is T; 864 is G;and 1016 is G; (c) a polypeptide having an amino acid sequence as setforth in SEQ ID NO:8 and having one or more amino acid modificationsselected from W68E, Q84W, A95P, K97C, S1168E, R181Y, N226C, Y277D or anycombination thereof, wherein the polypeptide has phytase activity; (d) apolypeptide having an amino acid sequence as set forth in SEQ ID NO:8and having the amino acid modifications W68E, Q84W, A95P, K97C, S168E,R181Y, N226C, Y277D, wherein the polypeptide has phytase activity; (e) apolypeptide encoded by a nucleic acid comprising a nucleotide sequenceas set forth in SEQ ID NO:1; (f) a polypeptide having an amino acidsequence as set forth in SEQ ID NO:2; or, (g) a combination of (a), (b),(c), (d), (e) or (f).
 32. A method for treating or preventingosteoporosis in an individual comprising administering to an individualan effective amount of a formulation as set forth in claim
 1. 33. Amethod for treating or preventing bone loss in an individual comprisingadministering to an individual an effective amount of a formulation asset forth in claim
 1. 34. A method for reversing bone loss orosteoporosis in an individual comprising administering to an individualan effective amount of a formulation as set forth in claim
 1. 35. Amethod for preventing muscle cramps comprising administering to anindividual an effective amount of a formulation as set forth in claim 1.36. A method for reducing pollution and increasing nutrient availabilityin an environment or environmental sample by degrading environmentalphytic acid comprising applying to the environmental or environmentalsample an effective amount of a composition comprising at least onepolypeptide having phytase activity, wherein the polypeptide comprises:(a) a polypeptide encoded by a nucleic acid comprising a nucleotidesequence as set forth in SEQ ID NO:7 and wherein nucleotide 389 is G;390 is A; 437 is T; 438 is G; 439 is G; 470 is C; 472 is T; 476 is T;477 is G; 478 is T; 689 is G; 690 is A; 691 is G; 728 is T; 729 is A;730 is T; 863 is T; 864 is G; or, 1016 is G; or, any combinationthereof, wherein the polynucleotide encodes a phytase; (b) a polypeptideencoded by a nucleic acid comprising a nucleotide sequence as set forthin SEQ ID NO:7 and wherein nucleotide 389 is G; 390 is A; 437 is T; 438is G; 439 is G; 470 is C; 472 is T; 476 is T; 477 is G; 478 is T; 689 isG; 690 is A; 691 is G; 728 is T; 729 is A; 730 is T; 863 is T; 864 is G;and 1016 is G; (c) a polypeptide having an amino acid sequence as setforth in SEQ ID NO:8 and having one or more amino acid modificationsselected from W68E, Q84W, A95P, K97C, S 168E, R181 Y, N226C, Y277D orany combination thereof, wherein the polypeptide has phytase activity;(d) a polypeptide having an amino acid sequence as set forth in SEQ IDNO:8 and having the amino acid modifications W68E, Q84W, A95P, K97C,S168E, R181 Y, N226C, Y277D, wherein the polypeptide has phytaseactivity; (e) a polypeptide encoded by a nucleic acid comprising anucleotide sequence as set forth in SEQ ID NO:1; (f) a polypeptidehaving an amino acid sequence as set forth in SEQ ID NO:2; or, (g) acombination of (a), (b), (c), (d), (e) or (f).
 37. The method of claim36, wherein the environment or environmental sample comprises a soil ora body of water.
 38. The method of claim 37, wherein the body of wateris well, a pond, a lake, a river, an aquifer or a reservoir.
 39. Themethod of claim 36, wherein the environment or environmental samplecomprises a sewage, a sewage effluent, a landfill or a manure pond.